Sox21 inhibits glioma progression in vivo by forming complexes with Sox2 and stimulating aberrant differentiation.

Sox2 is a transcription factor in neural stem cells and keeps the cells immature and proliferative. Sox2 is expressed in primary human glioma such as glioblastoma multiforme (GBM), primary glioma cells and glioma cell lines and is implicated in signaling pathways in glioma connected to malignancy. Sox21, the counteracting partner of Sox2, has the same expression pattern as Sox2 in glioma but in general induces opposite effects. In this study, Sox21 was overexpressed by using a tetracycline-regulated expression system (tet-on) in glioma cells. The glioma cells were injected subcutaneously into immunodeficient mice. The control tumors were highly proliferative, contained microvascular proliferation and large necrotic areas typical of human GBM. Induction of Sox21 in the tumor cells resulted in a significant smaller tumor size, and the effect correlated with the onset of treatment, where earlier treatment gave smaller tumors. Mice injected with glioma cells orthotopically into the brain survived significantly longer when Sox21 expression was induced. Tumors originating from glioma cells with an induced expression of Sox21 exhibited an increased formation of Sox2:Sox21 complexes and an upregulation of S100ß, CNPase and Tuj1. Sox21 appears to decrease the stem-like cell properties of the tumor cells and initiate aberrant differentiation of glioma cells in vivo. Taken together our results indicate that Sox21 can function as a tumor suppressor during gliomagenesis mediated by a shift in the balance between Sox2 and Sox21. The wide distribution of Sox2 and Sox21 in GBM makes the Sox2/Sox21 axis a very interesting target for novel therapy of gliomas.

Canine mammary tumors contain cancer stem-like cells and form spheroids with an embryonic stem cell signature.

We have investigated the presence of tentative stem-like cells in the canine mammary tumor cell line CMT-U229. This cell line is established from an atypical benign mixed mammary tumor, which has the property of forming duct-like structures in collagen gels. Stem cells in mammary glands are located in the epithelium; therefore we thought that the CMT-U229 cell line would be suitable for detection of tentative cancer stem-like cells. Side population (SP) analyses by flow cytometry were performed with cells that formed spheroids and with cells that did not. Flow cytometric, single sorted cells were expanded and re-cultured as spheroids. The spheroids were paraffin embedded and characterized by immunohistochemistry. SP analyses showed that spheroid forming cells (retenate) as well as single cells (filtrate) contained SP cells. Sca1 positive cells were single cell sorted and thereafter the SP population increased with repeated SP analyses. The SP cells were positively labeled with the cell surface-markers CD44 and CD49f (integrin alpha6); however the expression of CD24 was low or negative. The spheroids expressed the transcription factor and stem cell marker Sox2, as well as Oct4. Interestingly, only peripheral cells of the spheroids and single cells were positive for Oct4 expression. SP cells are suggested to correspond to stem cells and in this study, we have enriched for tentative tumor stem-like cells derived from a canine mammary tumor. All the used markers indicate that the studied CMT-U229 cell line contains SP cells, which in particular have cancer stem-like cell characteristics.

Forced expression of Sox21 inhibits Sox2 and induces apoptosis in human glioma cells.

Numerous studies support a role for Sox2 to keep stem cells and progenitor cells in an immature and proliferative state. Coexpression of Sox2 and GFAP has been found in regions of the adult brain where neural stem cells are present and in human glioma cells. In our study, we have investigated the roles of Sox2 and its counteracting partner Sox21 in human glioma cells. We show for the first time that Sox21 is expressed in both primary glioblastoma and in human glioma cell lines. We found that coexpression of Sox2, GFAP and Sox21 was mutually exclusive with expression of fibronectin. Our result suggests that glioma consists of at least two different cell populations: Sox2(+) /GFAP(+) /Sox21(+) /FN(-) and Sox2(-) /GFAP(-) /Sox21(-) /FN(+) . Reduction of Sox2 expression by using siRNA against Sox2 or by overexpressing Sox21 using a tetracycline-regulated expression system (Tet-on) caused decreased GFAP expression and a reduction in cell number due to induction of apoptosis. We suggest that Sox21 can negatively regulate Sox2 in glioma. Our findings imply that Sox2 and Sox21 may be interesting targets for the development of novel glioma therapy.

Sox10 has a broad expression pattern in gliomas and enhances platelet-derived growth factor-B--induced gliomagenesis.

In a previously published insertional mutagenesis screen for candidate brain tumor genes in the mouse using a Moloney mouse leukemia virus encoding platelet-derived growth factor (PDGF)-B, the Sox10 gene was tagged in five independent tumors. The proviral integrations suggest an enhancer effect on Sox10. All Moloney murine leukemia virus/PDGFB tumors had a high protein expression of Sox10 independently of malignant grade or tumor type. To investigate the role of Sox10 in gliomagenesis, we used the RCAS/tv-a mouse model in which the expression of retroviral-encoded genes can be directed to glial progenitor cells (Ntv-a mice). Both Ntv-a transgenic mice, wild-type, and Ntv-a p19Arf null mice were injected with RCAS-SOX10 alone or in combination with RCAS-PDGFB. Infection with RCAS-SOX10 alone did not induce any gliomas. Combined infection of RCAS-SOX10 and RCAS-PDGFB in wild-type Ntv-a mice yielded a tumor frequency of 12%, and in Ntv-a Arf-/- mice the tumor frequency was 30%. This indicates that Sox10 alone is not sufficient to induce gliomagenesis but acts synergistically with PDGFB in glioma development. All induced tumors displayed characteristics of PNET-like structures and oligodendroglioma. The tumors had a strong and widely distributed expression of Sox10 and PDGFR-alpha. We investigated the expression of Sox10 in other human tumors and in a number of gliomas. The Sox10 expression was restricted to gliomas and melanomas. All glioma types expressed Sox10, and tumors of low-grade glioma had a much broader distribution of Sox10 compared with high-grade gliomas.

Laminin alpha1 globular domains 4-5 induce fetal development but are not vital for embryonic basement membrane assembly.

During early mouse embryogenesis, each laminin (Lm) chain of the first described Lm, a heterotrimer of alpha1, beta1, and gamma1 chains (Lm-1), is essential for basement membrane (BM) assembly, which is required for pregastrulation development. Individual domains may have other functions, not necessarily structural. The cell binding C terminus of Lm alpha1 chain contains five Lm globular (LG) domains. In vitro, alpha1LG1-3 domains bind integrins, and alpha1LG4 binds dystroglycan, heparin, and sulfatides. A prevailing hypothesis is that alpha1LG4 is crucial as a structural domain for BM assembly, whereas integrin-binding sites conduct signaling. The in vivo role of alpha1LG4-5 (also called E3) has not been studied. Mice lacking alpha1LG4-5 were therefore made. Null embryos implanted, but presumptive epiblast cells failed to polarize and did not survive past day 6.5. BM components including truncated Lm alpha1 were detected in Reichert's membrane. Surprisingly, embryonic BM assembly between visceral endoderm and stem cells was normal in null embryos and in embryoid bodies of alpha1LG4-5-null embryonic stem cells. Yet, stem cells could not develop into polarized epiblast cells. Thus, alpha1LG4-5 provides vital signals for the conversion of stem cells to polarized epithelium.

Identification of candidate cancer-causing genes in mouse brain tumors by retroviral tagging.

Murine retroviruses may cause malignant tumors in mice by insertional mutagenesis of host genes. The use of retroviral tagging as a means of identifying cancer-causing genes has, however, almost entirely been restricted to hematopoietic tumors. The aim of this study was to develop a system allowing for the retroviral tagging of candidate genes in malignant brain tumors. Mouse gliomas were induced by a recombinant Moloney murine leukemia virus encoding platelet-derived growth factor (PDGF) B-chain. The underlying idea was that tumors evolve through a combination of PDGF-mediated autocrine growth stimulation and insertional mutagenesis of genes that cooperate with PDGF in gliomagenesis. Common insertion sites (loci that were tagged in more than one tumor) were identified by cloning and sequencing retroviral flanking segments, followed by blast searches of mouse genome databases. A number of candidate brain tumor loci (Btls) were identified. Several of these Btls correspond to known tumor-causing genes; these findings strongly support the underlying idea of our experimental approach. Other Btls harbor genes with a hitherto unproven role in transformation or oncogenesis. Our findings indicate that retroviral tagging with a growth factor-encoding virus may be a powerful means of identifying candidate tumor-causing genes in nonhematopoietic tumors.

Opposing roles of integrin alpha6Abeta1 and dystroglycan in laminin-mediated extracellular signal-regulated kinase activation.

Laminin-integrin interactions can in some settings activate the extracellular signal-regulated kinases (ERKs) but the control mechanisms are poorly understood. Herein, we studied ERK activation in response to two laminins isoforms (-1 and -10/11) in two epithelial cell lines. Both cell lines expressed beta1-containing integrins and dystroglycan but lacked integrin alpha6beta4. Antibody perturbation assays showed that both cell lines bound to laminin-10/11 via the alpha3beta1and alpha6beta1 integrins. Although laminin-10/11 was a stronger adhesion complex than laminin-1 for both cell lines, both laminins activated ERK in only one of the two cell lines. The ERK activation was mediated by integrin alpha6beta1 and not by alpha3beta1 or dystroglycan. Instead, we found that dystroglycan-binding domains of both laminin-1 and -10/11 suppressed integrin alpha6beta1-mediated ERK activation. Moreover, the responding cell line expressed the two integrin alpha6 splice variants, alpha6A and alpha6B, whereas the nonresponding cell line expressed only alpha6B. Furthermore, ERK activation was seen in cells transfected with the integrin alpha6A subunit, but not in alpha6B-transfected cells. We conclude that laminin-1 and -10/11 share the ability to induce ERK activation, that this is regulated by integrin alpha6Abeta1, and suggest a novel role for dystroglycan-binding laminin domains as suppressors of this activation.

Integrin alphavbeta3 binding to human alpha5-laminins facilitates FGF-2- and VEGF-induced proliferation of human ECV304 carcinoma cells.

Human ECV304 cells respond reproducibly by tube formation to complex basement membrane matrices. Laminins are major glycoproteins of basement membranes. We therefore studied the ability of ECV304 cells to attach to defined laminin isoforms and to fibronectin, and identified the involved laminin receptors. The cells bound poorly to fibronectin, to some extent to laminin-1, whereas laminin-2/4 and -10/11 were strong adhesive substrates. Antibody perturbation assays showed that adhesion to laminin-1 was mediated by integrin alpha6beta1, and adhesion to laminin-2/4 by cooperative activity of integrins alpha3beta1 and alpha6beta1. Adhesion of ECV 304 cells to laminin-10/11 was mainly mediated by integrins alpha3beta1, with minor involvement of alpha6beta1/4 and alphavbeta3. Solid-phase binding assays confirmed that integrin alphavbeta3 binds human laminin-10/11 and -10, in an RGD-dependent fashion. Although integrin alphavbeta3 played a very minor role in cell adhesion to laminin-10/11, this interaction facilitated growth factor-induced proliferation of ECV304 cells. In response to FGF-2 or VEGF, the cells proliferated better when attached on laminin-10/11 than on laminin-1, -2/4, or gelatin. The proliferation induced by the joint application of laminin-10/11 and either one of the growth factors could be blocked by antibodies against integrin alphavbeta3. Fragments of several other basement membrane components are known to interact with alphavbeta3. The current data show that that integrin alphavbeta3 can bind intact alpha5-containing laminin trimers. Since the laminin alpha5 chain is broadly expressed in adult basement membranes, this interaction could be physiologically important. Our data suggest that this interaction is involved in the regulation of cellular responses to growth factors known to be involved in epithelial and endothelial development.