Wnt signaling plays a substantial role in the control of development of several types of tissues through a dosage-dependent fashion. These regulated cells include crypt progenitor [28, 37], hair follicle , and hematopoietic stem cells . All these observations suggest that Wnt signaling is a dominant force in the control of proliferation of progenitor or stem cells. Our data further suggest that basic, moderate, physiological levels of Wnt signaling are sufficient for these cellular processes . To investigate the hypothesized role of Wnt signaling in the regulation of stemness networks in human cells, HONE1, SLMT1, and their hybrid cell lines were used. As expected, strong activation of endogenous tumor suppressors p53, RB1, and WT1 was detected in HONE1 hybrid cells, indicating that genomic integrity controlled by multiple tumor suppressor pathways within the parental HONE1 cells may be a key factor for maintaining a balanced environment to keep a low level of β-catenin expression [18–20].
Consistent with findings of both components of Wnt pathways and the core stem cell network being expressed or up-regulated in HONE1 hybrid cells, we found that the Wnt signal was clearly increased in HONE1 hybrid cells, as compared with untreated HONE1 cells. These in vitro observations were further confirmed in vivo with animal studies, indicating that there was consistent, up-regulated Wnt/β-catenin signaling in HONE1 hybrid cells.
To exclude the possibility that genes other than β-catenin on transferred chromosome 3 induced Wnt signaling in HONE1 hybrid cells, we specifically silenced β-catenin expression using β-catenin shRNA in these hybrid cells. Following the inhibition of β-catenin, expression of core stem cell genes and EMT markers was also decreased in the treated hybrid cells. These observations strongly indicate that β-catenin signaling, introduced by chromosome 3 transfer, is a dominant force in the regulation of both stemness and EMT networks in HONE1 cells, an expression pattern seen in other tissues [28, 30, 37–39].
Both chromosome 3 transfer and BIO treatment in HONE1 cells regulated EMT genes or its regulators. However, BIO only had similar, not identical biological effects as the chromosome 3 transfer, as evident in the control of cadherin switching. The Wnt signal controlling these EMT and cadherin networks was evidenced by the fact that β-catenin protein accumulated at the membranes, as seen in BIO-treated cells, hybrids with transferred chromosome 3, and recent stemness studies . This may be another regulatory process for entry control of free β-catenin into the nucleus, but the detailed mechanism of nuclear localization of β-catenin and the critical role E-cadherin plays in these processes are still not fully understood [8, 9, 21, 29, 41, 42].
The qPCR Array results confirmed that physiological β-catenin signaling triggered signals through additional pathways including pluripotency maintenance, FGF, and TGF-β superfamily signaling in HONE1 hybrid cells. For example, Smad2/5/9 and TGF-β receptors were clearly activated, which confirms previous reports that the TGF-β signaling pathway is associated with the pluripotency gene network and the EMT process [30, 41]. Many embryonic development genes such as Activin receptor, ACVR2A, were activated following the introduction of physiological β-catenin signaling in treated cells. This suggests that there are additional signals such as Activin that develop during culture and may serve to drive the reprogramming or cell self-renewal processes induced by Wnt signaling. It is also notable that the LIFR- and IL6ST-mediated pluripotency maintenance pathways, as well as BMP receptors and Smad families, were activated in HONE1 hybrid cells. Since these signals have well-known links to self-renewal programs, their increased expression provides additional evidence that physiological Wnt/β-catenin signaling is involved in the regulation of the cell self-renewal networks [4, 5, 8, 17, 43].
The stemness transition of HONE1 cells, driven by physiological Wnt/β-catenin signaling, appears to be a progressive process leading to a de-differentiated state in spheres, as demonstrated by analysis of up-regulated expression of core stem cell genes. Furthermore, genes that encode surface markers, such as CD9, CD24, CD44, CD90, and CD133, in spheres were expressed or clearly up-regulated compared to hybrid cells. Many Wnt signaling components in sphere-forming cells were identified as being involved in this stemness transition process, compared to hybrid cells. More than two-fold up-regulation of expression was detected in 34 genes, which included canonical, non-canonical, and Wnt/Ca+2 pathways.