APC dosage effects in tumorigenesis and stem cell differentiation

It is well established that concentration gradients of signaling molecules (the so-called "morphogens") organize and pattern tissues in developing animals. In particular, studies in Drosophila and different vertebrates have shown that gradients of the Wnt, Hedgehog (Hh) and transforming growth factor-beta (TGF-beta) families of morphogens play critical roles in limb patterning. Morphogens are often expressed in organizing centres and can act over a long range to coordinate the patterning of an entire field of cells. These observations imply that exposure to different concentrations of these diffusible factors may trigger differential cellular responses. In order to study these dosage-dependent Wnt/beta-catenin signaling effects, we have generated several hypomorphic mutant alleles at the mouse Apc locus and studied their cellular and phenotypic outcomes in stem cell renewal and differentiation, and in tumorigenesis. The results clearly show that Apc mutations differentially affect the capacity of stem cells to differentiate in a dosage-dependent fashion. Likewise, different Apc mutations (and the corresponding Wnt signaling dosages) confer different degrees of susceptibility to tumorigenesis in the corresponding mouse models. These results have implications for the understanding of the molecular and cellular basis of tumor initiation by defects in the Wnt pathway. We propose a model in which adult somatic stem cell compartments are characterized by tissue-specific beta-catenin threshold levels for cell proliferation, differentiation and apoptosis. Different APC mutations will result in different levels of beta-catenin signaling, thus conferring different degrees of tumor susceptibility in different tissues. Hence, beta-catenin dosage-dependent effects may not only explain how a single pathway is involved in the development and homeostasis of different tissues, but also its pleiotrophic role in tumorigenesis.     

Wnt activity and basal niche position sensitize intestinal stem and progenitor cells to DNA damage

Aging and carcinogenesis coincide with the accumulation of DNA damage and mutations in stem and progenitor cells. Molecular mechanisms that influence responses of stem and progenitor cells to DNA damage remain to be delineated. Here, we show that niche positioning and Wnt signaling activity modulate the sensitivity of intestinal stem and progenitor cells (ISPCs) to DNA damage. ISPCs at the crypt bottom with high Wnt/β‐catenin activity are more sensitive to DNA damage compared to ISPCs in position 4 with low Wnt activity. These differences are not induced by differences in cell cycle activity but relate to DNA damage‐dependent activation of Wnt signaling, which in turn amplifies DNA damage checkpoint activation. The study shows that instructed enhancement of Wnt signaling increases radio‐sensitivity of ISPCs, while inhibition of Wnt signaling decreases it. These results provide a proof of concept that cell intrinsic levels of Wnt signaling modulate the sensitivity of ISPCs to DNA damage and heterogeneity in Wnt activation in the stem cell niche contributes to the selection of ISPCs in the context of DNA damage.     

Wnt pathway mutations selected by optimal beta-catenin signaling for tumorigenesis

Mutations in components of the Wnt/beta-catenin pathway are observed to be the earliest initiating event for most colorectal tumors. The majority of the mutations occur in the tumor suppressor adenomatous polyposis coli (APC), even though there are other genes that are capable of modulating the pathway activity. Moreover, the specific APC mutations associated in colon cancer indicate the possibility that the tumor selects for certain truncated forms of APC that partially retain its function, namely, inhibition of beta-catenin. We estimated the effects of various mutations in APC and other known mutations using a recent mathematical model of the Wnt pathway that was constructed to represent the conserved core molecular events. We provide evidence that APC mutations are selected not based on the maximal level of beta-catenin but rather based on distinct state of activity that appears to be optimal for the tissue-specific tumorigenesis. This optimal level is determined by balancing beta-catenin signaling and the induction of Axin2 that acts as a potent negative feedback. The predominant pattern of APC mutations may provide synergistic oncogenic effects that promote colorectal tumorigenesis: the optimal signaling for cell survival and renewal, disrupted cell adhesion, chromosomal instability, and altered asymmetric division of stem cells.     

Nuclear beta-catenin localization is not sufficient for canonical Wnt signaling activation in human meianoma cell lines

In most cases, advanced stages of melanoma are practically incurable due to high metastatic potential of tumor cells. Multiple observations support the idea that aberrations in Wnt signaling pathway play a significant role in melanoma development and progression. Canonical Wnt signaling activation results in stabilization and accumulation of the major effector molecule called beta-catenin. Mutations promoting beta-catenin stabilization and, thereby, activation of canonical Wnt signaling pathway are frequently found in different cancers, but rarely observed in melanomas. Nevertheless, beta-catenin nuclear and cytoplasmic accumulation is the feature of many human melanoma cell lines and original tumors. That is why, the aim of the investigation was to elucidate the relation between beta-catenin intracellular localization and activity status of Wnt signaling pathway in human melanoma cell lines. Ten human melanoma cell lines were characterized on the basis of the following parameters: canonical Wnt ligand expression, intracellular beta-catenin localization, and activity status of canonical Wnt signaling pathway. Here, it has been demonstrated that nuclear localization of beta-catenin does not always correspond to active status canonical Wnt signaling pathway. Moreover, in the majority of cell lines with nuclear beta-catenin canonical Wnt signaling can't be activated by exogenous expression of an appropriate ligand. Human melanoma cell lines differ in activity of canonical Wnt signaling pathway as well as in mechanisms of its regulation. Therefore, the pathway-targeted potential antineoplastic therapy requires the formation of a "molecular pattern of cancer" for localization of the defect in Wnt signaling cascade in the each case.     

The stabilization of beta-catenin leads to impaired primordial germ cell development via aberrant cell cycle progression

Primordial germ cells (PGCs) are germ cell precursors that are committed to sperm or oocytes. Dramatic proliferation during PGC development determines the number of founder spermatogonia and oocytes. Although specified to a germ lineage, PGCs produce pluripotent embryonic germ (EG) cells in vitro and testicular teratomas in vivo. Wnt/beta-catenin signaling regulates pluripotency and differentiation in various stem cell systems, and dysregulation of this signaling causes various human cancers. Here, we examined the role of Wnt/beta-catenin signaling in PGC development. In normal PGC development, Wnt/beta-catenin signaling is suppressed by the GSK3beta-mediated active degradation of beta-catenin and the low expression of canonical Wnt molecules. The effects of aberrant activation of Wnt/beta-catenin signaling in PGCs were analyzed using mice carrying a deletion of the exon that encodes the GSK3beta phosphorylation sites in the beta-catenin locus. Despite the potential activity of Wnt/beta-catenin signaling in stem cell maintenance and carcinogenesis in various cell lineages, teratomas were not induced in the mice expressing the nuclear-localized beta-catenin in PGCs. Instead, the mutant mice showed germ cell deficiency caused by the delayed cell cycle progression of the proliferative phase PGCs. Our results show that the suppression of Wnt/beta-catenin signaling is a prerequisite for the normal development of PGCs.     

Beta-catenin up-regulates Nanog expression through interaction with Oct-3/4 in embryonic stem cells

It is well known that mouse embryonic stem (ES) cells can be maintained by the presence of leukemia inhibitory factor (LIF). Recent studies have revealed that Wnt also exhibits activity similar to LIF. The molecular mechanism behind the maintenance of ES cells by these factors, however, is not fully understood. In this study, we found that LIF enhances level of nuclear beta-catenin, a component of the Wnt signaling pathway. Expression of an activated mutant of beta-catenin led to the long-term proliferation of ES cells, even in the absence of LIF. Furthermore, it was found that beta-catenin up-regulates Nanog in an Oct-3/4-dependent manner and that beta-catenin physically associates with Oct-3/4. These results suggest that up-regulating Nanog through interaction with Oct-3/4 involves beta-catenin in the LIF- and Wnt-mediated maintenance of ES cell self-renewal.     

Wnt signaling controls the phosphorylation status of beta-catenin

At the heart of the canonical Wnt signaling cascade, adenomatous polyposis coli (APC), axin, and GSK3 constitute the so-called destruction complex, which controls the stability of beta-catenin. It is generally believed that four conserved Ser/Thr residues in the N terminus of beta-catenin are the pivotal targets for the constitutively active serine kinase GSK3. In cells that do not receive Wnt signals, glycogen synthase kinase (GSK) is presumed to phosphorylate beta-catenin, thus marking the latter for proteasomal degradation. Wnt signaling inhibits GSK3 activity. As a consequence, beta-catenin would no longer be phosphorylated and accumulate to form nuclear complexes with TCF/LEF factors. Although mutations in or near the N-terminal Ser/Thr residues stabilize beta-catenin in several types of cancer, the hypothesis that Wnt signaling controls phosphorylation of these residues remains unproven. We have generated a monoclonal antibody that recognizes an epitope containing two of the four residues when both are not phosphorylated. The epitope is generated upon Wnt signaling as well as upon pharmacological inhibition of GSK3 by lithium, providing formal proof for the regulated phosphorylation of the Ser/Thr residues of beta-catenin by Wnt signaling. Immunohistochemical analysis of mouse embryos utilizing the antibody visualizes sites that transduce Wnt signals through the canonical Wnt cascade.     

Negative Feedback Loop of Wnt Signaling through Upregulation of Conductin/Axin2 in Colorectal and Liver Tumors

Activation of Wnt signaling through beta-catenin/TCF complexes is a key event in the development of various tumors, in particular colorectal and liver tumors. Wnt signaling is controlled by the negative regulator conductin/axin2/axil, which induces degradation of beta-catenin by functional interaction with the tumor suppressor APC and the serine/threonine kinase GSK3beta. Here we show that conductin is upregulated in human tumors that are induced by beta-catenin/Wnt signaling, i.e., high levels of conductin protein and mRNA were found in colorectal and liver tumors but not in the corresponding normal tissues. In various other tumor types, conductin levels did not differ between tumor and normal tissue. Upregulation of conductin was also observed in the APC-deficient intestinal tumors of Min mice. Inhibition of Wnt signaling by a dominant-negative mutant of TCF downregulated conductin but not the related protein, axin, in DLD1 colorectal tumor cells. Conversely, activation of Wnt signaling by Wnt-1 or dishevelled increased conductin levels in MDA MB 231 and Neuro2A cells, respectively. In time course experiments, stabilization of beta-catenin preceded the upregulation of conductin by Wnt-1. These results demonstrate that conductin is a target of the Wnt signaling pathway. Upregulation of conductin may constitute a negative feedback loop that controls Wnt signaling activity.     

Wnt/beta-catenin signaling inhibits death receptor-mediated apoptosis and promotes invasive growth of HNSCC

The Wnt/beta-catenin signaling pathway plays a critical role in cell proliferation and oncogenesis. It has been found to be chronically activated in a variety of human cancers, including head and neck squamous cell carcinoma (HNSCC). Previously, we have found that the activation of the Wnt/beta-catenin signaling pathway inhibits mitochondria-mediated apoptosis. In this study, we extended our studies to determine whether the Wnt/beta-catenin signaling pathway inhibited death receptor-mediated apoptosis in HNSCC cells. We found that Wnt/beta-catenin inhibited not only tumor necrosis factor (TNF)/c-Myc-mediated apoptosis, but also cell detachment-mediated apoptosis (anoikis) which is dependent on the death receptor signaling pathway. Interestingly, we also observed that the Wnt/beta-catenin signaling pathway induced HNSCC cell scattering and promoted cell invasion in the Matrigel, both of which are hallmarks for the invasive growth of HNSCC. Consistently, the over-expression of beta-catenin promoted HNSCC tumor growth in nude mice. Taken together, our results suggest that the Wnt/beta-catenin signaling pathway plays dual functions in HNSCC development: promoting both cell survival and invasive growth of HNSCC cells.     

Defining early lineage specification of human embryonic stem cells by the orchestrated balance of canonical Wnt/beta-catenin, Activin/Nodal and BMP signaling

The canonical Wnt/beta-catenin signaling has remarkably diverse roles in embryonic development, stem cell self-renewal and cancer progression. Here, we show that stabilized expression of beta-catenin perturbed human embryonic stem (hES)-cell self-renewal, such that up to 80% of the hES cells developed into the primitive streak (PS)/mesoderm progenitors, reminiscent of early mammalian embryogenesis. The formation of the PS/mesoderm progenitors essentially depended on the cooperative action of beta-catenin together with Activin/Nodal and BMP signaling pathways. Intriguingly, blockade of BMP signaling completely abolished mesoderm generation, and induced a cell fate change towards the anterior PS progenitors. The PI3-kinase/Akt, but not MAPK, signaling pathway had a crucial role in the anterior PS specification, at least in part, by enhancing beta-catenin stability. In addition, Activin/Nodal and Wnt/beta-catenin signaling synergistically induced the generation and specification of the anterior PS/endoderm. Taken together, our findings clearly demonstrate that the orchestrated balance of Activin/Nodal and BMP signaling defines the cell fate of the nascent PS induced by canonical Wnt/beta-catenin signaling in hES cells.