| Background:Colon cancer is the common cause of cancer-related deaths worldwide. The success of therapy is severely hampered mainly due to the cancer metastasis and recurrence. However, the mechanism has not been addressed. Cancer stem cells (CSCs), a rare population in any type of cancers, including colon cancer, are tumorigenic. It has been thought that self-renewal and differentiation of CSCs are responsible for cancer recurrence, metastasis, and drug resistance. However, the CSCs are very rare, and in many cases, they cannot be detected. Thus, isolating CSCs in colon cancers is challenging and the molecular mechanism regulating the self-renewing and differentiation of CSCs remains unknown. CSCs in some cancers, including colon cancers, can be enriched by culturing cells in serum-free medium to form spheres. In this condition, Spheroid cells exhibit the common characteristics with CSCs. KLF4(Kruppel-like factor4) is a zinc finger transcription factor. It has been demonstrated that KLF4plays a role on maintaining the self-renewal of murine embryonic stem cells. Moreover, KLF4is also used to reprogram mouse fibroblasts to pluripotent stem cells, implying the role of K.LF4on maintaining the characteristics of stem cells. Importantly, the normal stem cells demonstrate common characteristics to cancer stem cells, it will also be important to determine whether similar regulation occurs in stem cells and cancer stem cells. It was reported that KLF4acts as an oncogene or tumor suppressor role in different tissue. Many groups reported that the KLF4is expressed and functions as a tumor suppressor in colon cancer. However, as reported, CSCs and the bulk of cancer cells are extremely heterogeneous and those studies did not distinguish colon CSCs from the bulk cancer cells. Moreover, CSCs are extremely rare, isolating CSCs becomes challenging. Thus, the expression and functions of KLF4in colon CSCs has not been reported. In our study, KLF4was decreased expressed in human colon cancer cell lines such as SW620, HT29, SW480, HCT116, DLD-1, LOVO compared to the human colon normal epithelium FHC cell line. Surprisingly, KLF4was increased in spheroid cells and CD133+subpopulation, which posses the characteristics of colon CSCs. As reported, KLF4plays the key roles in maintaining the characteristics of stem cells, and was overexpressed in colon CSCs enriched spheroid cells. Thus, whether KLF4functions as a tumor suppressor or as an oncogene to maintain the self renewal of colon CSCs need to be addressed.Objective:To investigate the expression of KLF4in colon cancer stem cells and KLF4in regulating the malignant biological behaviors in colon CSCs-enriched spheroid cells as well as to address KLF4in colon cancer metastasis and recurrence. It may provide new targets and ideas for the diagnosis and treatment of colon cancer.Methods:The expression levels of KLF4in SW620, HT29, SW480, HCT116, DLD-1, and LOVO human colon cancer cell lines as well as FHC, a normal colon epithelial cell line, were analyzed using real-time PCR and Western-blot. DLD-1cells were cultured in serum-free medium to form spheroid cells. For our convenient description, we designated the spheroid cells from DLD-1as DLD-S. The expression of core stem cell genes such as KLF4, Sox2, Oct4/3and Nanog, the expression of colon cancer stem cells genes such as CD133, CD166, Lgr5and ALDH1, as well as epithelial mesenchymal transition (EMT) related genes such as ZO-1, E-cadherin, Vimentin and Snail were analyzed by Real-time PCR and Western-blot, respectively. Moreover, immunofluorescence analysis confirmed the presence of E-cadherin and Vimentin. The migration and invasion of DLD-1and DLD-S cells were detected by transwell migration/invasion assay. The tumorigenic ability in vitro was confirmed by soft agar assay and colony formation assay. The tumorigenic ability in vivo was detected by subcutaneously injected cells into Balb/c nu/nu mice. The sensitivity of colon cancer cells to5-FU was analyzed using CCK-8assay kit. Furthermore, CD133+and CD133-cells were isolated from DLD-1cells by magnetic bead sorting using the MACS system. Subsequently, the expression of KLF4in the two subpopulations was demonstrated by Real-time PCR. The immunofluorescence analysis confirmed whether the co-staining of KLF4and CD133. The DLD-S siKLF4cells by infecting DLD-S cells with KLF4-shRNA lentiviral vector and the DLD-S siCon cells by infecting DLD-S cells with the non-target shRNA lentiviral vector were generated. The apoptosis of transfected cells was detected by Annexin-V APC/PI Double Labelling assay. The expression of KLF4, colon CSCs genes and EMT related genes were detected by real-time PCR and Western-blot. The capacities of migration, invasion and tumorigenicity in vitro and in vivo, as well as the sensitivity to5-FU were demonstrated as described. The capacity of self-renewal of those transfected cells was assessed by spheres-forming after culturing them in serum-free medium. Finally, the CD133+fraction was studied by cytometric analyses.Results:The expression of KLF4in various colon cancer cell lines were lower than the normal colon epithelial cell lines FHC (P<0.05). The core stem cell genes KLF4, Sox2, Oct4/3and Nanog as well as colon CSCs genes such as CD133, CD166, Lgr5, ALDH1were increased in DLD-S compared to DLD-1cells (P<0.05). Further study showed that the mRNA of KLF4was significantly higher in CD133+cells than in CD133-cells from the DLD-1cells (P<0.05). Expression of CD133and KLF4in DLD-S cells was further confirmed by immunofluorescent staining. Taken together, these results suggest that KLF4is most likely expressed in CSCs enriched populations. Moreover, DLD-S exhibited higher expression of epithelial gene E-cadherin, mesenchymal genes Snail and vimentin but lower of epithelial gene ZO-1, the key mediators of EMT (P<0.05). The capacities of migration, invasion and tumorigenicity in vitro and in vivo, as well as the sensitivity to5-FU were increased compared to the parent DLD-1cells (P<0.05). After generating DLD-S siKLF4cells and DLD-S siCon cells, the survival rate was similar among them as assessed by Annexin-V APC/PI Double Labelling assay (P>0.05). The expression of KLF4and colon CSCs genes were decreased in DLD-S siKLF4cells compared to DLD-S siCon cells (P<0.05). The number of CD133+cells is significantly lower in DLD-S siKLF4cells than in DLD-S siCon cells (P<0.05). Moreover, when cultured in serum-free medium, DLD-S siKLF4cells formed smaller spheres in a significantly lower frequency than DLD-S siCon cells did, suggesting that KLF4is essential for the self-renewal of DLD-S cells (P<0.05). At the same time, it is demonstrated that knockdown of KLF4expression in DLD-S cells crippled the capacities of these cells to migrate, invade, resist5-FU, and generate tumors in vitro and in vivo (P<0.05). Finally, knocking down KLF4expression in DLD-S cells decreased the expression of E-cadherin, Vimentin, and increased the expression of ZO-1(P<0.05).Conclusions:In summary, we successfully used serum-free culture system to enrich colon CSCs in most colon cell lines. Using these CSC-enriched spheroid cells, we conclude that KLF4functions as an oncogene for the development of colon cancer, challenging the previous conclusion of that KLF4functions as a tumor suppressor in colon cancer progression. Secondly, in our current studies, although KLF4induced expression of E-cadherin, consistent with the induced expression of vimentin and decreased expression of ZO-1as well as in vitro findings, it appears that KLF4induced EMT in colon CSCs-enriched population. Therefore, KLF4may be a potential target when we develop strategies to treat colon cancer. |
PDF Full Text Request