BACKGROUND
multiple groups have shown that different dosage levels of wnt signaling contribute to distinct cellular activities such as reprogramming, differentiation, tumorigenesis, and epithelial-mesenchymal transition  events  <cit> .
inappropriate activation of components of this signaling pathway has been observed in some human cancers and differentiating stem cells, in which high levels of wnt signaling were often detected  <cit> .
therefore, wnt signaling has multiple functions in cell fate determination and is involved in generation of cancer stem cells .
however, there are unresolved issues for the role of wnt/β-catenin signaling in the regulation of either self-renewal or differentiation networks in human cells  <cit> .
nasopharyngeal carcinoma  is a unique cancer, which is particularly prevalent among the southern chinese, but rare in most other areas around the world  <cit> .
unlike other common tumors, how wnt signaling influences npc and crosstalks to other networks to affect cell differentiation and growth, including regulation of csc markers and emt events, is unknown.
npc was reported to have an infrequent mutation of tumor suppressor gene  p <dig> and wild-type rb <dig> expression  <cit> ; they both play critical roles in the control of the reprogramming process, self-renewal, and other cell fate determinations  <cit> .
wnt signaling interacts with p <dig> signaling  <cit>  and usually acts in a dosage-dependent and tissue-specific manner for many cellular processes  <cit> .
therefore, it is possible to reveal novel findings by exploring the regulatory mechanism of wnt signaling in wild-type p <dig> expressing tumors such as with npc hone <dig> cells.
we previously established several microcell hybrid cell  lines derived from hone <dig> cells containing a transferred copy of chromosome  <dig>  <cit> .
because a physiological or basic level of wnt signaling acts as a determinant factor in the regulation of stem cells and self-renewing tissues  <cit>  and hone <dig> cells have very low endogenous expression of β-catenin, a major mediator of wnt signaling, we hypothesized that introduction of another copy of the β-catenin gene  via single copy transfer of chromosome  <dig> may generate physiological levels of β-catenin signaling due to the haploid level of expression of the transferred genes under control by their natural regulators.
this approach differs from gene transfection studies that often cause artificial overexpression of the transferred genes  <cit> .
in many cancer cells, stem cell-related genes are often expressed.
exogenous β-catenin signaling may regulate these endogenous signaling networks, thus changing the direction of cellular differentiation.
additionally, p <dig>  rb <dig>  or other possible tsgs, often serve as negative barriers for the reprogramming and self-renewal processes  <cit> .
delicate control of relevant signaling activities may drive cells into a more de-differentiated status, revealing signaling regulatory mechanisms during the stemness transition process, a series of regulatory relationships that are not fully understood in human cells.
it is important to determine what critical role β-catenin plays in the transferred chromosome by examining the relevant network activities in recipient cells.
it is well-accepted now that wnt/β-catenin signaling interacts with many other signaling networks such as pluripotency, cadherins, emt, transforming growth factor-β , fibroblast growth factor , and tsg signaling  <cit> .
if wnt/β-catenin signaling is activated, these relevant network activities are expected to be detected in treated cells.
for example, altered expression of e-cadherin and emt markers should be found in these cells.
therefore, whether wnt signaling, initiated at a basic and physiological level, is able to induce other signaling pathways during the progress of stemness transition, or to generate stem-like cells from human cancer cells, such as npc, is the focus of this study.
RESULTS
monochromosome  <dig> transfer confers physiological increases of β-catenin that up-regulates expression of core stem cell genes
we previously established several hone <dig> hybrid cell lines that were confirmed to contain an exogenous copy of the intact chromosome  <dig>  following fusion of parental hone <dig> and mouse mch <dig>  donor cells  <cit> .
figure 1a shows that both hone <dig> and mch <dig>  cells have similar and low expression levels of the human β-catenin, consistent with their having physiological levels of β-catenin signaling.
human embryonic stem cells, h <dig>  <cit> , were used as a positive control for mrna expression of stem cell genes and β-catenin.
the up-regulation of β-catenin expression was clearly detected in all three hone <dig> hybrid cell lines, as compared to hone <dig>  and is similar to that detected in h <dig> cells.
both c-myc and axin <dig> are major targets of the wnt pathway and tcf <dig> and tcf <dig> are terminal components of the β-catenin signaling pathway in the nucleus.
the expression of axin <dig> was detected in hone <dig> hybrid cells, but not in h <dig> cells and parental hone <dig> cells.
the expression of tcf <dig>  tcf <dig>  and c-myc were obviously up-regulated in these hone <dig> hybrid cells, compared with parental hone <dig> cells .
significant expression of pluripotency genes, nanog, oct <dig>  and klf <dig>  at the rna level was detected in hone <dig> hybrid cells compared to parental hone <dig> cells.
both hone <dig> and mouse mch <dig>  cells show endogenous expression of human sox <dig> gene, but in hone <dig> hybrid cells this gene was up-regulated and had similar expression levels as seen in h <dig> cells .
the increased level of β-catenin protein accumulating around membranes was clearly detected in the majority of hybrid cells compared to parental hone <dig> cells by immunofluorescence staining .
as expected, up-regulated protein levels of β-catenin, axin <dig>  nanog, and oct <dig> were detected by western blot analyses .
compared with parental hone <dig> cells, e-cadherin was up-regulated and n-cadherin was down-regulated in the hone <dig> hybrid cells.
enhanced wnt/tcf/lef network activity is maintained in vivo
a luciferase assay was performed using a wnt/tcf reporter plasmid that carries a large array of wnt response elements and is, therefore, a sensitive reporter of wnt signals.
as shown in figure 1d, transient transfection of the supertopgal reporter detected an increased level of wnt signaling by approximately 70-fold in hone <dig> hybrid cells.
immunohistochemical staining of the xenograft tumors demonstrated that β-catenin, e-cadherin, and cyclin d <dig>  the target of wnt signaling, were strongly up-regulated in tumor segregants  derived from hone <dig> hybrids, as compared with control tumors from the parental hone <dig> and mch <dig> -2ts cells.
both β-catenin and e-cadherin proteins clearly accumulated at the membrane , consistent with the pattern shown in figure 1b cultured cells.
it has long been known that some regions of transferred chromosomes in tss are selectively lost during tumor growth and the expression of relevant exogenous genes reverts back to levels similar to parental hone <dig> cells  <cit> .
xenograft tumors derived from hone <dig> hybrid cells were, therefore, analyzed for β-catenin expression .
for three of the hone <dig> hybrid tumor sets , increased β-catenin levels were evident as well as increased levels of endogenous stem cell genes nanog and oct <dig>  compared to corresponding hybrid cells.
a fourth hybrid cell line tumor, mch <dig> -2ts , did not show up-regulated expression of exogenous β-catenin and also did not have increased expression of nanog and oct <dig>
physiological expression of exogenous β-catenin is essential for the up-regulation of the endogenous core stem cell network
to further confirm whether expression of exogenous β-catenin is a major determinant in the regulation of the core stem cell network in hone <dig> hybrid cells, we performed inhibitory assays using short hairpin rnas  that silence β-catenin expression in infected cells.
a scrambled shrna was used as a negative control in all experiments.
hone <dig> hybrid cells with infected scramble shrna have up-regulated expression of β-catenin compared to parental hone <dig> cell .
hone <dig> hybrid cells with infected β-catenin shrna have reduced expression of β-catenin and representative results from various passages of cell populations after infection of shrna plasmids are shown in the bottom panel, figure 2a.
β-catenin expression was reduced by approximately 54%, 97%, 70%, and 79% in mch <dig>  and mch <dig>  cell lines at different passage levels after infection.
in these β-catenin shrna-infected cells, expression of nanog, oct <dig>  sox <dig>  and klf <dig> was inhibited as observed by qpcr analyses .
figure 2c shows that expression of β-catenin, e-cadherin, sox <dig>  zeb <dig>  and fn <dig> proteins were clearly decreased in both β-catenin shrna-treated cell lines.
the inhibited expression of oct <dig> was seen in mch <dig> , and inhibition of klf <dig> was detected in mch <dig> , but no obvious expression change was detected for n-cadherin and nanog.
these results suggest that β-catenin signaling in hone <dig> hybrid cells is involved in both stemness and emt networks.
hone <dig> hybrid cells are poorly differentiated and exhibit stem cell-like properties
parental hone <dig> cells grow as monolayers that do not form spheres under ordinary culture conditions, evidenced by our long-term culture of these cells  <cit> .
in contrast, after  <dig> to  <dig> days in standard culture,  <dig> spheres from independent experiments were observed in the hone <dig> hybrid cultures .
the size of all spheres was larger than  <dig> μm in diameters, which was not detected in parental hone <dig> and hone <dig> hybrid cell lines with infected β-catenin shrna.
these results suggest that sphere-forming cells had lost contact inhibition and had undergone a dedifferentiation process.
since csc markers, cd <dig> and cd <dig>  are commonly expressed in many stem-like cells, we investigated expression changes of these two markers by facs analysis.
cd24+, cd44+, and cd24+/cd44+ populations were markedly increased from 23% to  <dig> %,  <dig> % to  <dig> %, and  <dig> % to  <dig> %, respectively, in hone <dig> hybrid cells compared to parental hone <dig> cells .
physiological wnt levels trigger multiple signaling activities during the regulation of stem cell gene networks
the qpcr array results confirmed that pluripotency genes, sox <dig>  klf <dig>  oct <dig>  and nanog, are clearly up-regulated in hone <dig> hybrid cells, compared with parental hone <dig> cells.
in addition, many other relevant genes such as hoxa <dig>  rb <dig>  zic <dig>  wrn, tdgf <dig>  lin28b, and wt <dig> are considerably up-regulated in these hone <dig> hybrid cells , supporting the stem cell-like properties of these cells and activation of known genes related to self-renewal networks.
the exogenous copy of β-catenin in hone <dig> hybrid cells induced signaling cascades involved in multiple pathways such as wnt, tgf-β superfamily containing bmp and activin receptors, pluripotency maintenance, and fgf signaling .
for wnt signaling, enhanced expression of the non-canonical nfat family and wnt receptors, fzd <dig>  fzd <dig>  and fzd <dig>  was detected in these cells.
however, the stem cell signaling array did not detect significant changes of signals from hedgehog and notch pathways .
bio activation of wnt signaling generates similar signaling activities as induced by chromosome  <dig> transfer in hone <dig> cells
the gsk-3-specific inhibitor, bromoindirubin-3’-oxime , activates wnt signaling in stem cells and somatic cells  <cit>  and this small molecule was, therefore, used to treat parental hone <dig> cells to determine whether it had an effect on core stem gene signaling networks, similar to that seen in the hone <dig> hybrid cells.
as shown in figure 4c,  <dig> μm bio treatment induced the highest mrna expression of β-catenin and axin <dig>  bio treatment in hone <dig> cells up-regulated mrna expression of endogenous stem cell genes, nanog and oct <dig>  and emt markers and regulators such as twist, snail, cyclin d <dig>  slug, fn <dig>  and e-cadherin.
bio treatment also caused an up-regulation of n-cadherin in hone <dig> cells, suggesting that bio treatment does not fully mimic chromosome  <dig> transfer effects of inducing physiological signaling in hone <dig> cells.
bio-induced β-catenin accumulation was mainly detected at the membrane in treated cells .
to verify the critical role that physiological signaling levels exert, bio treatment was used to generate high, non-physiological levels of β-catenin protein expression in cells.
western blot analyses indicated that β-catenin, axin <dig>  and e-cadherin were strongly up-regulated in bio-treated hone <dig> cells.
these results were also confirmed in mch <dig> -2ts cells that lost a transferred copy of β-catenin gene.
importantly, endogenous protein levels of nanog and oct <dig> were not up-regulated in β-catenin over-expressing hone <dig> and mch <dig> -2ts cells .
chromosome  <dig> transfer does not affect cells with high levels of endogenous β-catenin expression
to determine whether physiological β-catenin signaling might regulate other human cells, esophageal carcinoma slmt <dig> cells were examined  <cit> .
the endogenous levels of β-catenin expression in slmt <dig> were higher than that of hone <dig> cells, likely due to an alternative transcript of β-catenin gene in exons 3/ <dig> in slmt <dig> cell observed by rt-pcr and sequencing analyses .
the expressions of both β-catenin and c-myc mrnas remain unchanged in slmt <dig> hybrid cells, mch <dig> / <dig>   <cit> , with an introduced copy of chromosome  <dig>  compared with their parental slmt <dig> cells .
nanog and oct <dig> protein expression was also unaltered in slmt1-derived hybrid cells .
tumor suppressor gene-mediated pathways are activated in hone <dig> hybrid cells
because the tumor suppressor p <dig> circuit communicates with the wnt pathway and serves as a major target in stem cells  <cit> , the expression of p <dig> in hone <dig> hybrid cells was examined at both the rna and protein levels.
interestingly, p <dig> was up-regulated and triggered by wnt signaling in all these hybrid cells by both rt-pcr  and western blot analyses .
as described previously, other tumor suppressors, such as rb <dig> and wt <dig> , were also activated in these cells, consistent with multiple tumor suppression pathways being activated following introduction of physiological wnt/β-catenin signaling in hone <dig> cells.
since tumor suppression pathways were activated in hone <dig> hybrid cells due to the up-regulation of wnt/β-catenin signaling, it may affect growth ability of cells.
to test these possibilities, mtt and colony formation assays in matrigel were performed with both stable scramble and β-catenin shrna-infected hone <dig> hybrid cells.
the results indicated that tumor cell growth was inhibited by the knockdown of wnt/β-catenin signaling in these cells , reflecting that activation of tumor suppressive signaling has limited influence to control cell growth in hone <dig> hybrid cells.
however, wnt/β-catenin signaling may stimulate cell growth during stemness transition process.
wnt signaling and stemness network activities are progressively up-regulated in sphere-forming cells
in the dedifferentiation progression of hone <dig> to hybrid to sphere cells, rna expression of four core stem cell genes, nanog, oct <dig>  sox <dig>  and klf <dig>  was continuously up-regulated in spheres compared to derived hybrid cells .
furthermore, some genes that encode csc surface markers  were either expressed or up-regulated by rt-pcr analysis in spheres, including cd <dig>  cd <dig>  cd <dig>  cd <dig>  and cd <dig>  notably, expression of genes that encode cd <dig> and cd <dig> was detected in sphere-forming cells derived from pooled large and small spheres from different experiments .
consistent with findings obtained from stem cell signaling array analysis , many wnt pathway components were activated, including both those in canonical and non-canonical signaling.
in spheres, lef <dig> was further increased 23-fold, compared to hybrid cells.
other markedly up-regulated genes are dkk <dig> , fzd <dig> , wnt <dig> , and fbxw <dig> .
additionally, expression of some components of wnt signaling, such as gsk3a  and the tle repressor aes , were clearly inhibited in these spheres, as seen in figures 7b and 7c.
discussion
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  <cit> , hair follicle  <cit> , and hematopoietic stem cells  <cit> .
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  <cit> .
to investigate the hypothesized role of wnt signaling in the regulation of stemness networks in human cells, hone <dig>  slmt <dig>  and their hybrid cell lines were used.
as expected, strong activation of endogenous tumor suppressors p <dig>  rb <dig>  and wt <dig> was detected in hone <dig> hybrid cells, indicating that genomic integrity controlled by multiple tumor suppressor pathways within the parental hone <dig> cells may be a key factor for maintaining a balanced environment to keep a low level of β-catenin expression  <cit> .
consistent with findings of both components of wnt pathways and the core stem cell network being expressed or up-regulated in hone <dig> hybrid cells, we found that the wnt signal was clearly increased in hone <dig> hybrid cells, as compared with untreated hone <dig> 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 hone <dig> hybrid cells.
to exclude the possibility that genes other than β-catenin on transferred chromosome  <dig> induced wnt signaling in hone <dig> 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  <dig> transfer, is a dominant force in the regulation of both stemness and emt networks in hone <dig> cells, an expression pattern seen in other tissues  <cit> .
both chromosome  <dig> transfer and bio treatment in hone <dig> cells regulated emt genes or its regulators.
however, bio only had similar, not identical biological effects as the chromosome  <dig> 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  <dig>  and recent stemness studies  <cit> .
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  <cit> .
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 hone <dig> hybrid cells.
for example, smad2/5/ <dig> 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  <cit> .
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 hone <dig> 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  <cit> .
the stemness transition of hone <dig> 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 cd <dig>  cd <dig>  cd <dig>  cd <dig>  and cd <dig>  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  <dig> genes, which included canonical, non-canonical, and wnt/ca+ <dig> pathways.
CONCLUSIONS
these studies demonstrate that both dosage levels of β-catenin signaling and genetic background of tissues play critical roles in the regulation of self-renewal networks in human cells.
there are very limited methods to explore physiological levels of wnt/β-catenin signaling in current biology studies and the results of these studies indicate that haploid levels of expression of transferred β-catenin can induce signaling cascades that include multiple known pathways associated with reprogramming and self-renewal processes.
appropriate wnt signaling may drive cancer cells into a progressive de-differentiated state, which generates expression profiles for exposure of the regulatory mechanism in stemness transition.
these novel results clarify the relationships that are still controversial and rather unclear at present among wnt signaling, stemness networks, emt events, tsg pathways and expression of common cancer stem cell surface markers.
these findings shed new light on a variety of cellular processes never before associated with physiological levels of β-catenin signaling that provide a framework for future studies to define the molecular mechanisms governing cell fate determination in human cells.

 multiple groups have shown that different dosage levels of wnt signaling contribute to distinct cellular activities such as reprogramming, differentiation, tumorigenesis, and epithelial-mesenchymal transition events. inappropriate activation of components of this signaling pathway has been observed in some human cancers and differentiating stem cells, in which high levels of wnt signaling were often detected. therefore, wnt signaling has multiple functions in cell fate determination and is involved in generation of cancer stem cells. however, there are unresolved issues for the role of wnt/β-catenin signaling in the regulation of either self-renewal or differentiation networks in human cells. nasopharyngeal carcinoma is a unique cancer, which is particularly prevalent among the southern chinese, but rare in most other areas around the world. unlike other common tumors, how wnt signaling influences npc and crosstalks to other networks to affect cell differentiation and growth, including regulation of csc markers and emt events, is unknown. npc was reported to have an infrequent mutation of tumor suppressor gene p and wild-type rb expression; they both play critical roles in the control of the reprogramming process, self-renewal, and other cell fate determinations. wnt signaling interacts with p signaling and usually acts in a dosage-dependent and tissue-specific manner for many cellular processes. therefore, it is possible to reveal novel findings by exploring the regulatory mechanism of wnt signaling in wild-type p expressing tumors such as with npc hone cells. we previously established several microcell hybrid cell lines derived from hone cells containing a transferred copy of chromosome. because a physiological or basic level of wnt signaling acts as a determinant factor in the regulation of stem cells and self-renewing tissues and hone cells have very low endogenous expression of β-catenin, a major mediator of wnt signaling, we hypothesized that introduction of another copy of the β-catenin gene via single copy transfer of chromosome may generate physiological levels of β-catenin signaling due to the haploid level of expression of the transferred genes under control by their natural regulators. this approach differs from gene transfection studies that often cause artificial overexpression of the transferred genes. in many cancer cells, stem cell-related genes are often expressed. exogenous β-catenin signaling may regulate these endogenous signaling networks, thus changing the direction of cellular differentiation. additionally, p rb or other possible tsgs, often serve as negative barriers for the reprogramming and self-renewal processes. delicate control of relevant signaling activities may drive cells into a more de-differentiated status, revealing signaling regulatory mechanisms during the stemness transition process, a series of regulatory relationships that are not fully understood in human cells. it is important to determine what critical role β-catenin plays in the transferred chromosome by examining the relevant network activities in recipient cells. it is well-accepted now that wnt/β-catenin signaling interacts with many other signaling networks such as pluripotency, cadherins, emt, transforming growth factor-β, fibroblast growth factor, and tsg signaling. if wnt/β-catenin signaling is activated, these relevant network activities are expected to be detected in treated cells. for example, altered expression of e-cadherin and emt markers should be found in these cells. therefore, whether wnt signaling, initiated at a basic and physiological level, is able to induce other signaling pathways during the progress of stemness transition, or to generate stem-like cells from human cancer cells, such as npc, is the focus of this study.  monochromosome transfer confers physiological increases of β-catenin that up-regulates expression of core stem cell genes we previously established several hone hybrid cell lines that were confirmed to contain an exogenous copy of the intact chromosome following fusion of parental hone and mouse mch donor cells. figure 1a shows that both hone and mch cells have similar and low expression levels of the human β-catenin, consistent with their having physiological levels of β-catenin signaling. human embryonic stem cells, h, were used as a positive control for mrna expression of stem cell genes and β-catenin. the up-regulation of β-catenin expression was clearly detected in all three hone hybrid cell lines, as compared to hone and is similar to that detected in h cells. both c-myc and axin are major targets of the wnt pathway and tcf and tcf are terminal components of the β-catenin signaling pathway in the nucleus. the expression of axin was detected in hone hybrid cells, but not in h cells and parental hone cells. the expression of tcf tcf and c-myc were obviously up-regulated in these hone hybrid cells, compared with parental hone cells. significant expression of pluripotency genes, nanog, oct and klf at the rna level was detected in hone hybrid cells compared to parental hone cells. both hone and mouse mch cells show endogenous expression of human sox gene, but in hone hybrid cells this gene was up-regulated and had similar expression levels as seen in h cells. the increased level of β-catenin protein accumulating around membranes was clearly detected in the majority of hybrid cells compared to parental hone cells by immunofluorescence staining. as expected, up-regulated protein levels of β-catenin, axin nanog, and oct were detected by western blot analyses. compared with parental hone cells, e-cadherin was up-regulated and n-cadherin was down-regulated in the hone hybrid cells. enhanced wnt/tcf/lef network activity is maintained in vivo a luciferase assay was performed using a wnt/tcf reporter plasmid that carries a large array of wnt response elements and is, therefore, a sensitive reporter of wnt signals. as shown in figure 1d, transient transfection of the supertopgal reporter detected an increased level of wnt signaling by approximately 70-fold in hone hybrid cells. immunohistochemical staining of the xenograft tumors demonstrated that β-catenin, e-cadherin, and cyclin d the target of wnt signaling, were strongly up-regulated in tumor segregants derived from hone hybrids, as compared with control tumors from the parental hone and mch -2ts cells. both β-catenin and e-cadherin proteins clearly accumulated at the membrane, consistent with the pattern shown in figure 1b cultured cells. it has long been known that some regions of transferred chromosomes in tss are selectively lost during tumor growth and the expression of relevant exogenous genes reverts back to levels similar to parental hone cells. xenograft tumors derived from hone hybrid cells were, therefore, analyzed for β-catenin expression. for three of the hone hybrid tumor sets, increased β-catenin levels were evident as well as increased levels of endogenous stem cell genes nanog and oct compared to corresponding hybrid cells. a fourth hybrid cell line tumor, mch -2ts, did not show up-regulated expression of exogenous β-catenin and also did not have increased expression of nanog and oct physiological expression of exogenous β-catenin is essential for the up-regulation of the endogenous core stem cell network to further confirm whether expression of exogenous β-catenin is a major determinant in the regulation of the core stem cell network in hone hybrid cells, we performed inhibitory assays using short hairpin rnas that silence β-catenin expression in infected cells. a scrambled shrna was used as a negative control in all experiments. hone hybrid cells with infected scramble shrna have up-regulated expression of β-catenin compared to parental hone cell. hone hybrid cells with infected β-catenin shrna have reduced expression of β-catenin and representative results from various passages of cell populations after infection of shrna plasmids are shown in the bottom panel, figure 2a. β-catenin expression was reduced by approximately 54%, 97%, 70%, and 79% in mch and mch cell lines at different passage levels after infection. in these β-catenin shrna-infected cells, expression of nanog, oct sox and klf was inhibited as observed by qpcr analyses. figure 2c shows that expression of β-catenin, e-cadherin, sox zeb and fn proteins were clearly decreased in both β-catenin shrna-treated cell lines. the inhibited expression of oct was seen in mch, and inhibition of klf was detected in mch, but no obvious expression change was detected for n-cadherin and nanog. these results suggest that β-catenin signaling in hone hybrid cells is involved in both stemness and emt networks. hone hybrid cells are poorly differentiated and exhibit stem cell-like properties parental hone cells grow as monolayers that do not form spheres under ordinary culture conditions, evidenced by our long-term culture of these cells. in contrast, after to days in standard culture, spheres from independent experiments were observed in the hone hybrid cultures. the size of all spheres was larger than μm in diameters, which was not detected in parental hone and hone hybrid cell lines with infected β-catenin shrna. these results suggest that sphere-forming cells had lost contact inhibition and had undergone a dedifferentiation process. since csc markers, cd and cd are commonly expressed in many stem-like cells, we investigated expression changes of these two markers by facs analysis. cd24+, cd44+, and cd24+/cd44+ populations were markedly increased from 23% to %, % to %, and % to %, respectively, in hone hybrid cells compared to parental hone cells. physiological wnt levels trigger multiple signaling activities during the regulation of stem cell gene networks the qpcr array results confirmed that pluripotency genes, sox klf oct and nanog, are clearly up-regulated in hone hybrid cells, compared with parental hone cells. in addition, many other relevant genes such as hoxa rb zic wrn, tdgf lin28b, and wt are considerably up-regulated in these hone hybrid cells, supporting the stem cell-like properties of these cells and activation of known genes related to self-renewal networks. the exogenous copy of β-catenin in hone hybrid cells induced signaling cascades involved in multiple pathways such as wnt, tgf-β superfamily containing bmp and activin receptors, pluripotency maintenance, and fgf signaling. for wnt signaling, enhanced expression of the non-canonical nfat family and wnt receptors, fzd fzd and fzd was detected in these cells. however, the stem cell signaling array did not detect significant changes of signals from hedgehog and notch pathways. bio activation of wnt signaling generates similar signaling activities as induced by chromosome transfer in hone cells the gsk-3-specific inhibitor, bromoindirubin-3’-oxime, activates wnt signaling in stem cells and somatic cells and this small molecule was, therefore, used to treat parental hone cells to determine whether it had an effect on core stem gene signaling networks, similar to that seen in the hone hybrid cells. as shown in figure 4c, μm bio treatment induced the highest mrna expression of β-catenin and axin bio treatment in hone cells up-regulated mrna expression of endogenous stem cell genes, nanog and oct and emt markers and regulators such as twist, snail, cyclin d slug, fn and e-cadherin. bio treatment also caused an up-regulation of n-cadherin in hone cells, suggesting that bio treatment does not fully mimic chromosome transfer effects of inducing physiological signaling in hone cells. bio-induced β-catenin accumulation was mainly detected at the membrane in treated cells. to verify the critical role that physiological signaling levels exert, bio treatment was used to generate high, non-physiological levels of β-catenin protein expression in cells. western blot analyses indicated that β-catenin, axin and e-cadherin were strongly up-regulated in bio-treated hone cells. these results were also confirmed in mch -2ts cells that lost a transferred copy of β-catenin gene. importantly, endogenous protein levels of nanog and oct were not up-regulated in β-catenin over-expressing hone and mch -2ts cells. chromosome transfer does not affect cells with high levels of endogenous β-catenin expression to determine whether physiological β-catenin signaling might regulate other human cells, esophageal carcinoma slmt cells were examined. the endogenous levels of β-catenin expression in slmt were higher than that of hone cells, likely due to an alternative transcript of β-catenin gene in exons 3/ in slmt cell observed by rt-pcr and sequencing analyses. the expressions of both β-catenin and c-myc mrnas remain unchanged in slmt hybrid cells, mch /, with an introduced copy of chromosome compared with their parental slmt cells. nanog and oct protein expression was also unaltered in slmt1-derived hybrid cells. tumor suppressor gene-mediated pathways are activated in hone hybrid cells because the tumor suppressor p circuit communicates with the wnt pathway and serves as a major target in stem cells, the expression of p in hone hybrid cells was examined at both the rna and protein levels. interestingly, p was up-regulated and triggered by wnt signaling in all these hybrid cells by both rt-pcr and western blot analyses. as described previously, other tumor suppressors, such as rb and wt, were also activated in these cells, consistent with multiple tumor suppression pathways being activated following introduction of physiological wnt/β-catenin signaling in hone cells. since tumor suppression pathways were activated in hone hybrid cells due to the up-regulation of wnt/β-catenin signaling, it may affect growth ability of cells. to test these possibilities, mtt and colony formation assays in matrigel were performed with both stable scramble and β-catenin shrna-infected hone hybrid cells. the results indicated that tumor cell growth was inhibited by the knockdown of wnt/β-catenin signaling in these cells, reflecting that activation of tumor suppressive signaling has limited influence to control cell growth in hone hybrid cells. however, wnt/β-catenin signaling may stimulate cell growth during stemness transition process. wnt signaling and stemness network activities are progressively up-regulated in sphere-forming cells in the dedifferentiation progression of hone to hybrid to sphere cells, rna expression of four core stem cell genes, nanog, oct sox and klf was continuously up-regulated in spheres compared to derived hybrid cells. furthermore, some genes that encode csc surface markers were either expressed or up-regulated by rt-pcr analysis in spheres, including cd cd cd cd and cd notably, expression of genes that encode cd and cd was detected in sphere-forming cells derived from pooled large and small spheres from different experiments. consistent with findings obtained from stem cell signaling array analysis, many wnt pathway components were activated, including both those in canonical and non-canonical signaling. in spheres, lef was further increased 23-fold, compared to hybrid cells. other markedly up-regulated genes are dkk, fzd, wnt, and fbxw. additionally, expression of some components of wnt signaling, such as gsk3a and the tle repressor aes, were clearly inhibited in these spheres, as seen in figures 7b and 7c. discussion 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, 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, hone slmt and their hybrid cell lines were used. as expected, strong activation of endogenous tumor suppressors p rb and wt was detected in hone hybrid cells, indicating that genomic integrity controlled by multiple tumor suppressor pathways within the parental hone cells may be a key factor for maintaining a balanced environment to keep a low level of β-catenin expression. consistent with findings of both components of wnt pathways and the core stem cell network being expressed or up-regulated in hone hybrid cells, we found that the wnt signal was clearly increased in hone hybrid cells, as compared with untreated hone 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 hone hybrid cells. to exclude the possibility that genes other than β-catenin on transferred chromosome induced wnt signaling in hone 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 transfer, is a dominant force in the regulation of both stemness and emt networks in hone cells, an expression pattern seen in other tissues. both chromosome transfer and bio treatment in hone cells regulated emt genes or its regulators. however, bio only had similar, not identical biological effects as the chromosome 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 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. 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 hone hybrid cells. for example, smad2/5/ 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. 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 hone 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. the stemness transition of hone 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 cd cd cd cd and cd 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 genes, which included canonical, non-canonical, and wnt/ca+ pathways.  these studies demonstrate that both dosage levels of β-catenin signaling and genetic background of tissues play critical roles in the regulation of self-renewal networks in human cells. there are very limited methods to explore physiological levels of wnt/β-catenin signaling in current biology studies and the results of these studies indicate that haploid levels of expression of transferred β-catenin can induce signaling cascades that include multiple known pathways associated with reprogramming and self-renewal processes. appropriate wnt signaling may drive cancer cells into a progressive de-differentiated state, which generates expression profiles for exposure of the regulatory mechanism in stemness transition. these novel results clarify the relationships that are still controversial and rather unclear at present among wnt signaling, stemness networks, emt events, tsg pathways and expression of common cancer stem cell surface markers. these findings shed new light on a variety of cellular processes never before associated with physiological levels of β-catenin signaling that provide a framework for future studies to define the molecular mechanisms governing cell fate determination in human cells. 
thirty-four up-regulated components of the wnt pathway were identified in these spheres. a few reports suggested that low levels of wnt signaling might drive cell reprogramming, but these studies could not establish a clear relationship between wnt signaling and self-renewal networks. qpcr array analyses further revealed interactions of physiological wnt/β-catenin signaling with other pathways such as epithelial-mesenchymal transition, tgf-β, activin, bmpr, fgfr and lifr- and il6st-mediated cell self-renewal networks. wnt/β-catenin signaling regulates self-renewal networks and plays a central role in the control of pluripotency genes, tumor suppressive pathways and expression of cancer stem cell markers. the nasopharyngeal carcinoma hone cells have low expression of β-catenin and wild-type expression of p which provided a possibility to study regulatory mechanism of stemness networks induced by physiological levels of wnt signaling in these cells. introduction of increased β-catenin signaling, haploid expression of β-catenin under control by its natural regulators in transferred chromosome resulted in activation of wnt/β-catenin networks and dedifferentiation in hone hybrid cell lines, but not in esophageal carcinoma slmt hybrid cells that had high levels of endogenous β-catenin expression. this current study provides a novel platform to investigate the interaction of physiological wnt/β-catenin signaling with stemness transition networks. physiological wnt/β-catenin signalingnasopharyngeal carcinomaself-renewal networkchromosome transferstemness transitiontumor suppressor genescancer stem cell markers additionally, whether physiological β-catenin signaling generates stem-like cells through interactions with other pathways is as yet unclear. hone hybrid cells displayed stem cell-like properties, including enhancement of cd24+ and cd44+ populations and generation of spheres that were not observed in parental hone cells.
 a few reports suggested that low levels of wnt signaling might drive cell reprogramming, but these studies could not establish a clear relationship between wnt signaling and self-renewal networks. there are ongoing debates as to whether and how the wnt/β-catenin signaling is involved in the control of pluripotency gene networks. additionally, whether physiological β-catenin signaling generates stem-like cells through interactions with other pathways is as yet unclear. the nasopharyngeal carcinoma hone cells have low expression of β-catenin and wild-type expression of p which provided a possibility to study regulatory mechanism of stemness networks induced by physiological levels of wnt signaling in these cells.  introduction of increased β-catenin signaling, haploid expression of β-catenin under control by its natural regulators in transferred chromosome resulted in activation of wnt/β-catenin networks and dedifferentiation in hone hybrid cell lines, but not in esophageal carcinoma slmt hybrid cells that had high levels of endogenous β-catenin expression. hone hybrid cells displayed stem cell-like properties, including enhancement of cd24+ and cd44+ populations and generation of spheres that were not observed in parental hone cells. signaling cascades were detected in hone hybrid cells, including activation of p53- and rb1-mediated tumor suppressor pathways, up-regulation of nanog-, oct4-, sox2-, and klf4-mediated pluripotency networks, and altered e-cadherin expression in both in vitro and in vivo assays. qpcr array analyses further revealed interactions of physiological wnt/β-catenin signaling with other pathways such as epithelial-mesenchymal transition, tgf-β, activin, bmpr, fgfr and lifr- and il6st-mediated cell self-renewal networks. using β-catenin shrna inhibitory assays, a dominant role for β-catenin in these cellular network activities was observed. the expression of cell surface markers such as cd cd cd cd and cd in generated spheres was progressively up-regulated compared to hone hybrid cells. thirty-four up-regulated components of the wnt pathway were identified in these spheres.  wnt/β-catenin signaling regulates self-renewal networks and plays a central role in the control of pluripotency genes, tumor suppressive pathways and expression of cancer stem cell markers. this current study provides a novel platform to investigate the interaction of physiological wnt/β-catenin signaling with stemness transition networks. physiological wnt/β-catenin signalingnasopharyngeal carcinomaself-renewal networkchromosome transferstemness transitiontumor suppressor genescancer stem cell markers 