Differential Connexin Function Enhances Self-Renewal in Glioblastoma

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1 CSU ID: Name: Sonal Patel Differential Connexin Function Enhances Self-Renewal in Glioblastoma Cancer is one of the most common disease in the US with more than a million people affected every year. Numerous theories have been postulated to understand its cause and mechanisms, yet, with no complete cure for the disease. Several studies of the cancerous tumors have revealed that it is a chunk of heterogeneous population of cells. Previously, these cells were thought to be evolved through the clonal evolution theory where a cell undergoes series of mutation and gives rise to several of its kind and others that have similar tumorigenic potential(nowell, Peter 1976). Later in 1990s, a new theory of cancer stem cell drew large attention of many researchers(bonnet & Dick 1997; Lapidot et al. 1994). According to the Cancer Stem Cell (CSC) theory, there are a few stem cells-like cells, capable of self-renewing, that have tumorigenic capacity and are able to sustain the tumor through generation of heterogeneous population of cells. Multiple pathways could give rise to the tumor cells either from a single or multiple CSCs. Also not all types of cancers consist of CSCs as the tumor initiating cells while others such as glioblastomas (GBM) contain several self-renewing CSCs that contribute to tumor formation and propagation. Few tumors consist of biologically distinct population of CSCs such as that observed in PTEN- deficient glioblastoma(chen et al. 2010). Glioblastoma is one of the fatal and most common type of a brain tumor. Therapeutic resistance and tumor heterogeneity are the major hurdles that make the current therapies ineffective in treatment of this tumor. GBM tumor cells is known to consist of a large number of CSCs that add to the therapeutic resistance. One of the biggest barrier in understanding the biology of a tumor is the heterogeneous population of the cells with the CSCs contributing majorly. CSCs play most critical role in tumor development and are the major threat in the treatment of the disease due to its mysterious behavior. During the treatment these cells could enter the latent state with the

2 dangerously high risk of relapse. Also as a part of protection mechanism CSCs could revert back to non-cscs and vice versa, which could again make their detection difficult. CSCs survival largely depends on its microenvironment including growth factors, nutrients and communication with the adjacent cells(visvader & Lindeman 2012). In 1966, Loewenstein and colleagues, first implicated the role of gap junctions in tumorigenesis and since then it has been a target for many researchers(loewenstein & Kanno 1966). Gap junctions are defined as the cell to cell junctions that holds the plasma membranes in close apposition and allows for the passage of nutrients, signals and other metabolites from one cell to another. The gap is bridged by 21 connexin proteins that form channels to allow substances to pass through. These have tissue specific expression and functions in development, differentiation and metabolic homeostasis. Its dysfunction is related to connexin-type specific diseases such as cataract (Cx 46 and Cx 50)(Beyer & Berthoud 2014). Connexins were hypothesized to have a role in tumor suppression since several studies in rodent tumors and tumor cell lines have demonstrated the downregulation of connexin proteins. Also, connexin knockout mice are more prone to tumor formation when challenged with carcinogenic agents(naus & Laird 2010). In accordance with the tumor suppressive role, Cx43, one of the most widely studied connexin is known to be downregulated in GBM while its overexpression in CSCs is related to tumor latency(yu et al. 2012). However, recent data shows a more complicated role of connexins in facilitating tumor progression as opposed to suppression. Hitomi et al paper demonstrate that connexin, Cx46, is required for tumor progression in GBM. Further, it is shown that Cx46 is upregulated in CSCs and is required for the growth and maintenance of the tumor. Xenograft models T4121 and T387 which are the classical and mesenchymal subtypes have been used in this study. Figure 1 shows the presence of the gap junctions in the xenograft tumor sections and CSC spheres invitro by electron microscopy. CSCs were isolated using papain dissociation test and identified using stem cell specific markers, CD133 and α6 integrin. The functional cell-cell communication through gap junction was

3 confirmed by microinjecting the dye along with the fluorescent IgG such that the dye diffused easily through the gap junctions while the IgG being too large in size was not able to pass through, as observed by the time lapse imaging. Further the functional role of gap junctions was confirmed by similar single cell injection of the dye particles in the presence of several gap junction inhibitors such as carbenoxolone (CBX) and 1-octanol etc. As observed in figure 2, the diffusion of dye through the gap junctions is significantly reduced in CSCs isolated from both the xenograft models. Since the CSCs seem to have functional gap junctions, it would be interesting to study the effect of blocking these gap junctions on the phenotype of CSCs. To study this, growth of CSCs was checked in the presence of gap junction inhibitors. As expected, the growth of CSCs dramatically reduced upon inhibiting gap junctions (Figure 3A). Further, self-renewal ability of the CSCs was checked by sphere formation assay using range of concentrations of inhibitors. Reduction in the number of spheres formed per well as well as stem cell frequency was observed (Figure 3B and C). The reduction in the growth of CSCs was due to increased apoptosis as was observed by the increased caspase3/7 activity. Also the specificity of the inhibitor towards CSCs population was confirmed by comparing the IC50 value of CSCs vs NPCs. The effect of gap junction inhibitors on tumor growth was assayed in the presence of anti-tumor agent, temozolomide (TMZ). Inhibitor alone was able to reduce the tumor volume significantly whereas additive effect on tumor growth suppression was observed when combined with TMZ. Interestingly, the effect was also seen in orthotopic tumor model as the mice with intracranial tumors given gap junction inhibitor for 14 days demonstrated increased tumor latency as well as survival. Furthermore, gap junction inhibitors decreased the growth of the TMZ-resistant population sensitizing a large population of tumor cells to TMZ thus increasing its efficacy. After establishing the role of gap junctions in the maintenance of CSCs in GBM tumor next step was to find which connexins aid in tumor formation and maintenance. For this qpcr analysis was carried out to study the expression of different connexins in the NPCs and GBM models. Analysis

4 showed Cx46 expression to be significantly elevated in CSCs while Cx43 was high in non-csc population. It was confirmed by immunoblot analysis of Cx46 which showed enrichment in CD133+ CSC cells than in non-cscs where Cx43 levels were found to be higher. Location of Cx46 and Cx43 was evaluated by differentiating the CSCs using differentiation medium with 10% serum and removal of growth factors. Upon differentiation Cx46 levels reduced while Cx43 levels increased in agreement with the above results. Thus Cx46 is highly expressed in CSCs while Cx43 in non CSCs which increases upon differentiation of CSCs. To understand the role of Cx46 in CSCs, its self-renewal and proliferation ability was tested in the absence of Cx46. Therefore, Cx46 was knockdown (KD) in CSCs using shrna constructs. Upon Cx46 KD, growth of CSCs was stunted with induced apoptosis as assessed by caspase activity along with reduction in its self-renewal ability and stem cell frequency. If Cx46 was required for tumor formation was further assessed by injecting Cx46 KD CSCs into the brains of immunocompromised mice. Upon Cx46 KD, mice demonstrated high tumor latency and reduced tumor forming capacity compared to the controls which suggests that Cx46 is necessary for tumor formation and maintenance of CSC population in GBM tumor. Next question was, what is the physiological consequence of Cx46 high expression in CSCs? What is its effect on the resting membrane potential in CSCs? To answer these questions dye diffusion method was used to check for the relative diffusion between the CSCs upon Cx46 KD. New gap junction establishment assay showed that CSCs were quick in establishing new gap junctions compared to non CSCs as is shown in Figure 5. Also diffusion was relatively slower in Cx46 KD CSCs compared to the normal CSCs. Moreover outward current flow measured in CSCs was rather reduced than in non CSCs possibly due to inhibition of flow of potassium across the membranes which was also confirmed by the depolarized resting membrane potential in CSCs. Based on previous studies Cx43 and Cx46 expressions are known to be linked. Indeed, Cx43 KD in non CSCs leads to increased expression of Cx46 in CSCs which is in correlation with the increased resting membrane potential observed in Cx43 KD as in normal CSCs. This implicates

5 that the expression of different connexins in CSCs and non CSCs dictates differentiation that in turn affects gap junction formation and resting membrane potential. To establish the relation between Cx46 and GBM patient survival, bioinformatics analysis was carried out of data available in several datasets such as Oncomine and the National Cancer Institute (NCI) Repository for Molecular Brain Neoplasia Data (REMBRANDT). Analysis revealed poor prognosis of GBM and glioma patients upon Cx46 upregulation as is presented in Figure 6. However, no such correlation was observed between different other connexins and patient survival indicating the importance of Cx46 in CSC maintenance. Overall, although the connexins, as gap junction proteins, have been well established as exhibiting anti-tumor effect as its reduced levels correlate with the increased incidences of tumors, this paper talks about the pro tumorigenic role of one of the connexin, Cx46. Though the link between Cx43 and Cx46 has been shown in the paper it requires further detailed analysis as there could be several important proteins such as those involved in EMT, cadherins, linking these two major proteins. A study in Cx46 Knockouts and tumor development in these animals would provide further insights into the mechanism and role of Cx46 in CSCs maintenance. Another question that needs to be addressed is; what will be the effect of knockdown of both Cx46 and Cx43 in tumor formation and CSCs? Also the inhibitors used in this study target several connexins and are not specific for Cx46 thus a connexin specific inhibitor should be tested. It is also necessary to test the resistance to drugs other than TMZ since the effect of gap junctions to see if the increased efficacy is universal for GBM drugs or it is drug specific effect. However, this paper highlights an important point relating to the isotype specific role of connexins. Hence, development of therapies targeting gap junction proteins should be carefully considered, as not all proteins involved in gap junctions behave in a similar manner or have similar functions. Nevertheless, this paper adds many more questions to the GBM horizon that could contribute in unravelling the cause of this mysterious disease.

6 Beyer, E.C. & Berthoud, V.M., Connexin hemichannels in the lens. Frontiers in Physiology, 5 FEB. Bonnet, D. & Dick, J.E., Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nature Med., 3, pp Available at: Chen, R. et al., A Hierarchy of Self-Renewing Tumor-Initiating Cell Types in Glioblastoma. Cancer Cell, 17(4), pp Lapidot, T. et al., A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature, 367(6464), pp Loewenstein, W.R. & Kanno, Y., Intercellular communication and the control of tissue growth: lack of communication between cancer cells. Nature, 209(5029), pp Naus, C.C. & Laird, D.W., Implications and challenges of connexin connections to cancer. Nature reviews. Cancer, 10(6), pp Nowell, Peter, C., The Clonal Evolution of Tumor Cell Populations. Science. Visvader, J.E. & Lindeman, G.J., Cancer stem cells: Current status and evolving complexities. Cell Stem Cell, 10(6), pp Yu, S.C. et al., Connexin 43 reverses malignant phenotypes of glioma stem cells by modulating E-cadherin. Stem Cells, 30(2), pp

CRIPTO-1 A POSSIBLE NEW BIOMARKER IN GLIOBLASTOMA MULTIFORME PIA OLESEN, MD, PHD STUDENT

CRIPTO-1 A POSSIBLE NEW BIOMARKER IN GLIOBLASTOMA MULTIFORME PIA OLESEN, MD, PHD STUDENT Glioblastoma WHO Grade IV Glioma Heterogenic Undiffenrentiated phenotype 50% of all Gliomas Around 600 patients