The present study indicates that a reduction in SFRP1 expression allows 76 N TERT cells to acquire many of the properties observed in breast cancer cells. These SFRP1 knocked down cells (TERT-siSFRP1) have increased levels of β-catenin in their nucleus, which is indicative of β-catenin stabilization and activated Wnt/β-catenin signaling. Moreover, TERT-siSFRP1 cells undergo a dramatic phenotypic change which can be explained in part by a genotypic change that is consistent with EMT. Similar to malignant mammary cells, TERT-siSFRP1 cells are more resistant to anchorage-independent cell death, are quite migratory and invasive, and exhibit a CD44high/CD24low cell surface marker expression pattern. Finally, microarray analysis revealed that the characteristics exhibited by TERT-siSFRP1 are consistent with affected pathways including cell death, cell-to-cell signaling, and interaction cellular assembly.
The downstream effector of Wnt signaling, β-catenin, exists in three different subcellular forms: membrane-bound (complexed with E-cadherin), cytosolic, and nuclear . The data presented here show that β-catenin accumulates in the nucleus of TERT-siSFRP1 cells where it is available to act as a transcription factor. Considering that genes regulated by β-catenin are involved in tumorigenesis, the nuclear accumulation of β-catenin may be a possible mechanism by which SFRP1 renders the mammary gland more susceptible to tumorigenesis. Interestingly, Nakopoulou et. al. have demonstrated that increased nuclear staining for phospho-β-catenin in breast tumor specimens is associated with poorer overall survival (OS). Similarly, Veeck et. al. have found increased promoter hypermethylation of SFRP1 is also associated with poor prognosis . Putatively, SFRP1 loss may be associated with nuclear β-catenin accumulation in human breast tumors. Interestingly, β-catenin-mediated transcription from an exogenous reporter construct was not activated unless exogenous Wnt3a was added to the media. These findings suggest that either greater amounts of nuclear β-catenin are necessary to see significant differences in reporter activity or that SFRP1 loss is affecting alternate pathways through either non-canonical Wnt signaling or Wnt-independent mechanisms.
Previously, Shulewitz et. al. ultilized siRNA oligonucleotides to transiently silence SFRP1 MCF-10A breast epithelial cells. In order to evaluate the permanent changes in cellular behavior that occur in response to SFRP1 down-regulation, we chose to create the TERT-siSFRP1 stable cell line to . Our results are consistent with Shulewitz et. al. in that TERT-siSFRP1 cells exhibit an increase in β-catenin-mediated luciferase activity and CyclinD1 expression in response to Wnt3a stimulation. CyclinD1 is a cell-cycle regulator essential for G1 phase progression  and the pro-proliferative nature of this oncogene is implicated in pathogenesis of several human tumor types, including breast carcinomas . Our findings clearly demonstrate that the increase in Cyclin D1 expression, due to aberrant β-catenin transcriptional activation, observed in TERT-siSFRP1 cells contributes to an increase in cellular proliferation. However, other proliferative cues must be affected by SFRP1 loss because the enhanced cellular proliferation occurs both in the presence as well as the absence of the Wnt3a ligand whereas Cyclin D1 expression is increased only when Wnt3a is added to TERT-siSFRP1 cells.
Tumor cell metastasis occurs when primary epithelial cells exit their site of origin and colonize at a distant site. In order for this process to occur, the cells must invade the extracellular matrix, migrate into blood vessels, and invade secondary organs. Here we show that when SFRP1 is down-regulated, 76 N TERT cells become significantly more migratory as well as invasive. The phenotypic changes observed when SFRP1 is down-regulated include a transformation from an epithelial cell morphology to a more fibroblast-like cell with reduced intercellular adhesion and increased motility. Interestingly, these characteristics define the EMT process, which is known to occur during tumor progression as well as metastasis. After careful assessment of several genes implicated in EMT, we can conclude that loss of SFRP1 expression likely pushes 76 N TERT cells into this pathophysiological transformation.
E-cadherin down-regulation is frequently associated with Wnt signaling and we show here that E-cadherin is significantly repressed in TERT-siSFRP1 cells. Loss of E-cadherin expression is emerging as one of the most common indicators of EMT onset, as it normally acts as a tumor suppressor by inhibiting invasion [47, 48]. Considering that both ZEB1 and ZEB2 are key transcriptional repressors of E-cadherin, and the expression levels of ZEB1 and ZEB2 are drastically enhanced in response to SFRP1 down-regulation, it is reasonable to suspect that the loss in E-cadherin expression is a direct result of the increase in these transcription factors. The expression of vimentin is also frequently associated with the EMT process and is significantly upregulated in TERT-siSFRP1 cells. The increase in vimentin expression may be due to the ~100-fold increase in ZEB2 expression, as ZEB2 is also involved in up-regulating the expression of vimentin in breast cancer cells . Interestingly, Gilles et. al. have shown that β-catenin can transactivate vimentin expression which could explain, in part, the increase in vimentin expression when SFRP1 is down-regulated .
It is well established that de-regulated expression and altered function of the genes involved in cell-cycle regulation and death contribute to the pathogenesis of cancer  and SFRP1 exhibits an apoptotic function in several different tissues, including breast epithelial cells [52–54]. However, the data presented here are the first to show that SFRP1 down regulation renders mammary epithelial cells resistant to cell death. Moreover, this characteristic is observed in anchorage-independent growth conditions and in the absence of exogenous Wnt3A ligand. Interestingly, caspase-1 is significantly down-regulated in TERT-siSFRP1 cells (data not shown). Although the role of caspase-1 down regulation has not been studied in breast cancer cells, it is lost in other cancer cell lines [55, 56]. The microarray data provide some other suggestions as to how TERT-siSFRP1 cells become resistant to anoikis, such as the increase in the pro-survival gene, Mdm-2, which is a potent regulator of p53. We also noted an increase in ATRX, a member of the SWI/SNF family of chromatin remodeling proteins, which has been previously shown to be involved in neuronal cell survival . However, further work is required to fully elucidate the mechanisms by which SFRP1 loss increases anchorage independent cell survival.
Metastatic breast cancer cells are often characterized by a CD44high/CD24low cell surface marker expression pattern . Although it is logical that cells that have undergone EMT and exhibit this cell surface marker expression pattern are able to break out from the primary tumor and metastasize to distant locations, it has remained poorly understood as to how such cells could self-renew to form secondary metastases. Recent work by Mani et al. has addressed this dilemma by genetically modifying non-malignant mammary epithelial cells to undergo EMT . Not surprisingly, these cells were shown to exhibit a CD44high/CD24low phenotype. Although this cell surface marker expression pattern is associated with metastatic potential, it is also observed in both normal as well as breast cancer stem cells . Forced EMT allowed the cells to exhibit additional characteristics associated with progenitor cells including an increased ability to form primary as well as secondary mammospheres, differentiate into secondary structures, and express both luminal and basal/myoepithelial markers, which exemplifies the bipotency of these cells . We have observed that loss of SFRP1 also results in primary and secondary mammosphere formation (data not shown) and we are currently investigating whether these cells have additional stem cell-like characteristics.
It is noteworthy to point out that non-conical Wnt signaling is also known to induce cell migratory phenotypes during developmentand isoften separated into two subcategories including the Planar Cell Polarity (PCP) pathway and the Wnt/Ca2+ pathway . The EMT associated phenotype observed in TERT-siSFRP1 cells may be explained in part by particular sections of the signal transduction cascades that characterize these pathways. First, PCP signals through Rho and Rac GTPases to alter cell polarization and affect the actin cytoskeleton resulting in an increase in cellular motility and directional migration. Second, the Wnt/Ca2+ pathway can activate the GTPase CDC42, which forms a complex with Par3 and atypical protein kinase, and this pathway has been previously implicated in ErbB2 mediated EMT in mammary epithelial cells . Finally, the up-regulation of TMSB4X, filamin beta, and ADAMTS15 noted in the microarray analysis support the pro-migratory and invasive features and hint at the possible role of either other non-canonical Wnt pathways and/or Wnt-independent EMT mechanisms also being induced by SFRP1 loss [59–61].
The reduction in SFRP1 could potentially alter other pathways, independent of Wnt signaling, which could contribute to a malignant transformation and progenitor cell phenotype. For instance, Notch signaling may very well be aberrantly activated in TERT-siSFRP1 cells and exacerbate their aggressive phenotype. Ayyanan et. al. previously showed that Wnt-1 treated HMECs undergo a malignant transformation which is a result of not only activated β-catenin, but also activated Notch signaling [62, 63]. Our data lends support to the hypothesis that the loss of SFRP1 may facilitate Notch signaling. Specifically, TERT-siSFRP1 cells have increased levels of the APH1A gene, which is a member of the gamma-secretase complex required for the intramembrane proteolysis of and activation of Notch. Furthermore, Mdm-2 is an ubiquitin ligase which targets the Notch inhibitor, Numb, for degradation . Lastly, Notch is frequently activated in CD44hi/CD24lo breast epithelial cells and inhibition of gamma-secretase activity has been shown to block the ability of the cells to form mammospheres and sensitizes the cancer cells to undergo growth arrest and apoptosis [65, 66]. Research is ongoing to determine the importance of Notch signaling in the physiological characteristics imparted by SFRP1 loss.