Direct Contact By Melanoma Cells Causes Malignant Transformation Of Human Epithelial-like Stem Cells Via Alpha V Integrin Activation Of TGF-β1 Signaling

Direct contact by melanoma cells causes malignant transformation of human epithelial-like stem cells via alpha V integrin activation of TGF-β1 signalingBackground and objectiveThe somatic mutation theory of cancer has been the prevailing paradigm in cancer research for decades and states that malignant transformation is the result of accumulation of multiple mutations in normal differentiated cells. Recent studies have demonstrated that not all tumor cells have the capacity to perpetuate the tumor, and that only a subset of tumor cells with stem cell-like properties, called cancer stem cells (CSCs), is capable of driving tumor initiation, progression, and recurrence. The current CSC hypothesis suggests CSCs, which arise from normal stem cells (SCs) by mutation of genes that make the stem cells cancerous, is consistent with the classical model of multi-step carcinogenesis, as SCs have longer life spans than their differentiated progeny and could accumulate multiple mutations over many years.Somatic SCs reside in a specialized physical local known as a'niche'.The microenvironment in the niche is essential for the maintenance and functioning of normal SCs. According to the CSC hypothesis, the first step of tumor formation is the clonal expansion of a mutation-harboring SC within a niche, known as pretumor progression. With additional mutations, these cells may expand beyond the confines of an individual niche into microscopic or larger sized patches/field. For instance, colon tumors are thought to progress through at least five stages:pretumor patches/fields, hyperplasia, carcinoma in situ, invasive carcinoma and metastasis.The microenvironment has major effects on the development and progression of cancer. In the classic two-stage tumorigenesis model, the microenvironment has been proposed to play a part in the promotion phase. Several groups have demonstrated the involvement of microenvironmental factors in tumor progression and proposed that the microenvironment may promote cancer progression through distinct signaling mechanisms. For example, Olumi reported that carcinoma-associated fibroblasts can direct tumor progression of initiated prostate epithelial cells by SV40-T.Recent attention has been given to the interactions between stem cells and melanoma cells. Indeed, the bi-directional microenvironmental communication may be propagated among cells through at least three different mechanisms:direct cell-cell contact, autocrine and paracrine signaling driven by soluble secreted factors, and modeling (or re-modeling) of the extracellular matrix. Postovit and colleagues studied the epigenetic influence of the microenvironment of human embryonic stem cells on the reprogramming of aggressive melanoma cells using an innovative 3D culture model. They demonstrated redifferentiation of the melanoma cells to a melanocyte-like phenotype after exposure to the microenvironment of human embryonic stem cells. Further studies with this model revealed that the aggressive melanoma-conditioned matrix microenvironment induced transdifferentiation of normal melanocytes into melanoma-like cells exhibiting a vasculogenic phenotype. These studies demonstrated that the embryonic microenvironment may have a significant influence on the phenotypic characteristics of aggressive cancer cells; however, the effects of a tumorigenic microenvironment on normal stem cells have not been sufficiently explored.In this study, we established a novel in vitro 3D co-culture model system in order to induce malignant transformation of human epithelial-like stem cells (hEpSCs) in a microenvironment created by the A375 melanoma celi line. Using a 2-step protocol (exposure to the mutagen 7,12-dimethylbenz[a]anthracene (DMBA) and subsequent direct contact with A375 melanoma cells), we showed that hEpSCs could be converted to melanoma cells via a mechanism involving integrin alpha V-mediated activation of TGF-β1 signaling.Part I hEpSCs undergo maligant transformation and epithelial-mesenchymal transition when treated with DMBA and then cultured in contact with A375 melanoma cells.Methods:(1) To characterize the cells:Using immunofluorescence methods to detectβ1-integrin, K19 and p63 in the 4-8 passage HEMSC. (2) Establish in vitro co-culture model of A375 and hEpSCs:hEpSCs were treated with 0.1μg/ml DMBA (Sigma-Aldrich) for 16 h prior to the co-culture with A375 cells. Three groups of cultures were studied:1) Contact Group:hEpSCs or hEpSCs treated with DMBA+A375 melanoma cells in contact co-culture by seeding on the bottom and top sides of porous membrane of a cell culture insert, respectively; 2) No Contact Group:hEpSCs or hEpSCs treated with DMBA+A375 melanoma cells co-cultured without contact (separated by cell culture insert) but allowing hEpSCs to be exposed to the A375 conditioned media; and,3) Control (control cultures containing hEpSCs or hEpSCs treated with DMBA only). (3) To assess the tumorigenesis of transformed hEpSCs, we established a xenograft model.hEpSCs at passage 6, which were treated with DMBA or not treated with DMBA and then were induced by A375 for 7days, were suspended in DMEM before implantation to facilitate establishment. (4) Transformational ability of hEpSCs after co-culture:Using immunofluorescence methods and flow Cytometry analysis to detect N-cahherin and Vimentin in the in 7d of co-cultured hEpSCs. In 3,5,7d, E-cahherin protein were assayed by western blotting respectively.Results:To assess the tumorigenesis of hEpSCs or hEpSCs treated with DMBA in the three groups, we established a xenograft model. Fourteeen days after receiving hEpSCs or hEpSCs treated with DMBA in the three groups, nodules appeared in nude mice and only hEpSCs treated with DMBA in the contact group fcrmed black nodules and therefore, the cells we studied in the following experiments were hEpSCs treated with DMBA and A375 cells. The growth kinetics of hEpSCs treated with DMBA in the contact group xenografts in nude mice (n=6 for each experimental group) showed the proliferous ability of transformed hEpSCs in vivo. HE staining of the subcutaneous tissue showed significant differences among 3 experimental groups. Fontana-Masson staining for the melanin revealed hyper-pigmentation in the contact group, but not in the other two groups. However, hEpSCs without treated with DMBA in the three groups of cultures presented no pigmentation in the contact group. HLA-1 immunohistochemistry confirmed the human origin of the cells with hyper-pigmentation. These data indicated that transformed hEpSCs were able to form melanoma in vivo.Transformed hEpSCs colonies were observed at d7 in hEpSCs treated with DMBA with direct contact to A375 cells, but not in the non-contact group or hEpSCs treated with DMBA cultured alone. The "pile-up" appearance of hEpSCs colonies in the contact group prompted us to examine their anchorage-independent growth in soft agar. We carefully wiped A375 cells on the bottom of porous membrane of the cell culture using a cotton swab, digested hEpSCs on the top sides of the cell culture insert with 0.25% trypsin and seeded these cells in the three groups separately in soft agar at the density of 1000 cells per well. After incubation for 3 weeks, in contrast to that no colonies were found in control cells, nearly 5% of hEpSCs in the contact group formed large colonies, indicating that hEpSCs with direct contact to A375 cells had underwent the transformation. Additionally, we monitored the growth curve for hEpSCs in the three groups and showed that hEpSCs with direct contact to A375 cells had the enhanced proliferation prominently in vitro. Reduction of E-cadherin and concomitant production of N-cadherin are features of epithelial-mesenchymal transition (EMT). In the hEpSCs treated with DMBA and then induced by A375 cells, expression of N-cadherin and Vimentin was significantly increased, whereas expression of E-cadherin was decreased in the same experiment group. As is known, down-regulation of E-cadherin is the hallmark molecular change that occurs during EMT. The number of cells positive for N-cadherin and Vimentin in the contact group was significantly increased. Since N-cadherin and Vimentin are mesenchymal markers, these results clearly indicated that transformed hEpSCs possess mesenchymal characteristics and EMT. However, hEpSCs without treated with DMBA in the three groups of cultures presented no phenotype changes and transformation.Part II Malignant transformation of human epithelial-like stem cells via alpha V integrin activation of TGF-β1 signalingMethods:(1) The aforementioned observations indicate that malignant transformation of the hEpSCs required direct contact with melanoma. We speculate that direct contact between melanoma cells and hEpSCs may stimulate certain paracrine signals. To identify these signals, in vitro co-cultures of A375 cells and hEpSCs were established and their conditioned media were measured for the levels of various cytokines and growth factors by ELISA. (2) To detect phospho-Smad2 (p-Smad2), a downstream signal transduction factor of TGF-β1 in A375 and hEpSCs contact co-culture group and hEpSCs alone culture group were made as in Part One. In 0,6,12, 24,48h, p-Smad2 in hEpSCs were detected by western blotting; while p-Smad2 by immunocytochemistry method in the meantime. (3) hEpSCs (5×106) at passage 6, which were treated with DMBA and then were induced by A375 for 7days, were suspended in DMEM before implantation(100μL) to facilitate establishment. (4) To further test the accumulation of ITGAV in the transformed hEpSCs, mRNA expression of ITGAV was assessed by RT-PCR. To explore.the role of ITGAV in the process of tumorigenesis, we established stable siRNA expression vectors, pGCsilencerTM U6/NEGative (NCRNAi) and pGCsilencerTM U6/ITGAV (ITGAV RNAi). (5) To investigate the functional consequences of ITGAV interference in the process of tumorigenesis, the growth kinetics of transformed hEpSCs with ITGAV interference in vivo were compared to those of hEpSCs over 4 weeks. (6) Since ITGAV and TGF-β1 were involved in the transformation of hEpSCs, we next set out to resolve the relationship between ITGAV and TGF-β1. We measured the levels of TGF-β1 in the media of hEpSCs before and after stable knockdown of 90% ITGAV.Results:(1) In some cases, certain released factors (for example, MMP-1 or Gro) were higher in the contact group than in the remaining two groups. Notably, TGF-β1 was 3-fold higher in the contact culture group than the hEpSCs alone or no-contact co-culture group, suggesting a synergistic interaction between A375 and hEpSCs. This cooperative induction of TGF-β1 was apparent as early as the third day of co-culture. To determine whether TGF-β1 signal pathways in hEpSCs are activated in the contact co-culture group, we measured the phospho-Smad2 (p-Smad2), a downstream signal transduction factor of TGF-β1. Importantly, subsequent co-culture of these hEpSCs and cancer cells continued to allow accumulation of p-Smad2 in hEpSCs in the contact co-culture group that were comparable to those observed in the no-contact group and control group. Meanwhile, immunocytochemistry revealed that p-Smad2 could be detected in the nuclei of 30-50% transformed hEpSCs, suggesting that direct contact between melanoma cells and hEpSCs activates the TGF-β1/Smad signaling pathways. (2) mRNA expression of ITGAV was increased in contact co-culture. To explore the role of ITGAV in the process of tumorigenesis, we established stable siRNA expression vectors, pGCsilencerTM U6/NEGative (NCRNAi) and pGCsilencerTM U6/ITGAV (ITGAV RNAi). After transfection of NCRNAi and ITGAV RNAi in cultured hEpSCs, colonies (NC, A2, C1, D2 and F4) were selected by neomycin (200ug/mL). Western blots revealed that ITGAV RNAi treatment decreased total cellular ITGAV protein levels. Histograms analysis showed the average level of ITGAV interference was 50%,30%,70% and 90% for A2, Cl, D2 and F4, respectively. (3) ITGAV interference inhibited the tumor growth. More importantly, ITGAV with interference efficiency of 90% and 70% of hEpSCs, their tumor volume displayed a significant decrease. These data suggested that ITGAV may contribute to the tumorigenesis. ELISA analysis revealed that stable knockdown of 90% ITGAV did not result in accumulated levels of TGF-β1 in the contact groups. Western blots of P-Smad2 in hEpSCs after the difference knockdown ITGAV levels in contact co-culture group at 3d also showed a weakening of TGF-(31 signaling. Taken together, these observations suggested that ITGAV mediate the local activation of TGF-β1/Smad signaling pathways.PartⅢTransformed hEpSCs demonstrate impaired polarityMethods:(1) To determine whether polarity in transformed hEpSCs has changed, we performed intracellular expression studies for integrin alpha V (ITGAV), Par polarity proteins and Tiam1 by triplicate immunostaining and Western blot. (2) To determine whether polarity in transformed hEpSCs after stable knockdown of 90% ITGAV has changed, we performed intracellular expression studies for integrin alpha V (ITGAV), Par polarity proteins and Tiaml by triplicate immunostaining and Western blot.Results:(1) ITGAV was expressed in the cytoplasm; immunoreactive signals were significantly increased in transformed hEpSCs. Expression of endogenous Par3 protein and TIAM1 were consistently decreased at the leading edge and cytoplasm of hEpSCs, but not at leading edges of transformed hEpSCs. Endogenous p-aPKCζwere asymmetrically enriched throughout the hEpSCs in transformed hEpSCs, expression of endogenous p-aPKCζwas decreased in the nucleus. Western blot analysis of ITGAV, TIAM1, p-aPKCζand PAR3 confirmed these findings, suggesting that polarity of transformed HEpSCs had been disrupted. (2) Expression of endogenous Par3 protein and TIAM1 were also consistently decreased at the leading edge and cytoplasm of hEpSCs after stable knockdown, but not at leading edges of transformed hEpSCs. Endogenous p-aPKCζwere asymmetrically enriched throughout the hEpSCs in transformed hEpSCs after stable knockdown, expression of endogenous p-aPKCζwas decreased in the nucleus. Western blot analysis of ITGAV, TIAM1, p-aPKCζand PAR3 confirmed these findings, suggesting that polarity of transformed HEpSCs had been disrupted. We proved that TIAM1, p-aPKCζand PAR3 may be the hallmark of malignant transformation of hEpSCs.Conclusions: (1) We successfully established a model in vitro for inducing malignant transformation of epithelial-like stem cells with melanoma cell. The data have proved that epidermal-like stem cell could obtain the associated characteristics of tumor cell by the inducement of A375.This suggests that self-renewing and differentiation of stem cell in the tumor microenvironment be out of control.(2) Since DMBA treated hEpSCs by the inducement of A375 showed tumorigenesis in vivo, this data suggests hEpSCs have obtained neoplastic transformation.(3) These data suggest that the microenvironment of metastatic melanoma cells could lead to cancer formation of normal stem cell and imply that stem cells must be the targets of carcinogenic exposure. They also help to better understand the origins of malignant stem cells and their responses to to be critica to the therapy of malignancies.