t Role of Dlxin-1 in cell proliferation and tumorigenecity in Neuro - epithelial cell-lin< • H I t « I t N o 4^ Introduction Recent studies focus on cancer as a stem cell disorder exhibiting altered stem- cell maturation/differentiation programs with disturbances in regenerative processes. Many malignancies seem to follow the hierarchical model of cancer development, wherein so called cancer stem cells or cancer-initiating cells, are wholly responsible for the continued grov\rth and propagation of the tumor. These tumor initiating subset of cells with 'stem like' characteristics and capability for tumor initiation is reported for a range of solid tumors that include colon, breast and brain tumors. Glioma is one of the most common type of primary brain tumor (Kleihues, P. et a!., 2000). Recent, studies have demonstrated the existence of a small fraction of glioma cells with a tumor-initiating function, endowed with features of primitive neural progenitor cells (Hemmati HD et al., 2003). Cellular origins and genetic factors governing the genesis and maintenance of glioblastomas (GBM) are still not very well understood. Dlxin-1 is broadly expressed during development and in vitro studies have implicated Dlxin-1 in cellular functions ranging from cell cycle regulation, cell adhesion to transcriptional regulation (Xue B. et al., 2005; Wen,C.J. et al., 2004; Zhou LH, 2006; Masuda et al., 2001). It is now known that endogenous NRAGE cooperates with Necdin to promote terminal differentiation of post mitotic myoblasts and neuroblasts by repressing cell proliferation (Kuwajima et al., 2004). NRAGE is a versatile pro-apoptotic protein required for neuronal apoptosis that is regulated by the p75 neurotrophin receptor (Bertrand, M. J. et al., 2008). ^ 1) ^ 33 Dlxin-1 is an important regulator of cellular differentiation and proliferation in the Central Nervous System (CNS). MAGED1 RNA is highly expressed in the fetal and adult brain with highest expression detected in the cerebral cortex (Bertrand M et al., 2004), while its expression is found low in gliomas. Recent studies have demonstrated the existence of a small fraction of glioma cells endowed with features of primitive neural 100 progenitor cells with a tumor-initiating function that is mainly reported in the CD133 positive fraction (Dirks PB et al., 2008). It is not known whether, the tumor initiating stem cells, show a differential expression of Dlxin-1 compared to other glioma cells that are not enriched for stem cell features. While, there are reports that Dlxin-1 is as an anti- tumorigenic and anti-invasive protein in pancreatic cancer, melanoma (Chu, C. S. et al., 2007) and in breast cancer cell-lines (Tian et al., 2005), its expression and function in cancer stem cells is completely unknown. Cancer cell lines serve as alternative sources of CSCs. Recently, CSCs have been identified from a number of glioma cell-lines like U87MG (Yu SC et al., 2008) and in C6 gliomas {In Vitro, 2008). We have earlier reported on development of an in vitro model to understand Glioblastoma progression comprising 2 cell-lines HNGC-1 and HNGC-2 (Shiras et al., 2007). The HNGC-2 cell are enriched with a high percentage of CD133 positive cells, when grown as Neurospheres, and is an attractive model to investigate the role of Dlxin-1 on glioma stem cells. In this study, we have exploited this model to get an insight on the role of Dlxin-1 on cancer stem cells. We over-expressed Dlxin-1 in glioma cells (LN18) as well as in glioma cancer stem cells (HNGC-2), to specifically determine if Dlxin is effective as an anti-proliferative and anti- tumorigenic protein in the CD133 positive, drug resistant glioma stem cells and to know its role in self-renewal, proliferation, tumorigenesis and differentiation of glioma stem cells. In this chapter, we showed that, Dlxin-1 besides exerting a strong anti­ proliferative, anti-tumorigenic and anti-invasive response on glioma stem cells induced profound changes in the sternness signature of glioma stem cells. Dlxin-1, increased self-renewal potential of cancer stem cells and induced their conversion to a neural stem cell like state. Taken together, our work highlights on the relevance of Dlxin-1 as a therapeutic target for glioma stem cells with profound implications in designing molecular strategies targeted towards the drug resistant CSCs. Results 3. Role of Dlxin-1 In cell proliferation and tumorigenecity in Neuro-epithelial cell- lines 3. 1. Lower Levels of Dlxin-1 mRNA in Glioma Cell Lines Compared with Primary brain. Since, Dlxin-1 is shown to be expressed in the human brain we analyzed and compared the mRNA expression profiles of Dlxin-1 in normal brain tissue, with human established glioma cell-lines (Neuro-epithelial cell-lines). While, the human universal cDNA used as a positive control and fetal and adult brain tissues expressed high levels of Dlxin-1, its expression in glioma cell-lines, U87MG, LN229 and LN18 though variable was quite low (Fig. 1A). Strikingly, the glioma cancer stem cell-line HNGC-2 showed the lowest Dlxin- 1 expression. Over-expression of Dlxin-1 cDNA and HA-tagged Dlxin-1 cDNA into cell- lines LN18 and HNGC-2 generated stable cell-lines LN18-Dlxin-1 and HNGC-2-Dlxin-1 respectively, that over-expressed the transcript (Fig. 1A) and HA-tagged Dlxin-1 protein as shown in Fig1B. / DAPI ,/ B. Antibody MwBMl / ' //7/////// LN18-HA-Dlxln-1 1 i Olxin-1 HNGC2-HA-DlKin-1 AeUn ^3 • " " 1 1 Fig. 3.1. Dlxin-1 Expression In human brain and glioma cell-lines. A. Tiie e^^»^^^^ Dixin-1 was analyzed by RT-PCR using human universal, fetal brain and adult whole brain cDNAs and from cDNAs of established human glioma cell lines U87MG, LN229, LN18, cancer stem cells - HNGC-2 and from ceils over-expressing Dlxin-1 i.e.- LN18- Dixin-1 and HNGC-2-Dixin-1, using gene specific primers. The human universal cDNA and cDNAs derived from fetal brain and adult whole brain were from Cionetech, whereas all the other cDNAs used in this study are from RNA, extracted from cells, grown in complete medium, p-actin served as an internal control. B. Dlxin-1 obtained as HA- tagged protein was transfected into both LN-18 and HNGC-2 cells. Stable independent clones of both LN-18 and HNGC-2 over-expressing Dlxin-1 were generated. The Cell-lines LN18-Dlxin-1 and HNGC-2-Dixin-1 represent three pools of stable independent transfectants. These transfectant cells were confirmed for Dlxin-1 (green) over- expression by Immuno-fluorescence assays using antibody to HA-protein. Nuclei were stained with DAPI (blue). Scale - lOum. 102 3. 2. Growth Characteristics of Dixin-1 over-expressing cells Accumulating evidences indicate that gliomas arise due to a small fraction of tumor-initiating precursors with stem-like properties known as cancer stem cells. Similar to normal neural stem cells, the brain tumor initiating cells (BTICs), display self-renewal potential in vitro, form clonal Neurospheres and are multipotent. The BTICs have the potential to establish GBMs at the clonal level and in most cases are CD133 positive. These BTSCs are refractory to the current cytotoxic therapies and hence identification of novel strategies that can directly target BTSCs would have immense therapeutic potential. From a glioma tumor tissue, we have developed a long term in vitro culture HNGC-2, that can be propagated as xenografts in immuno-compromised mice, wherein the cells in low numbers form intra-cranial tumors resembling GBM. The HNGC-2 cells satisfy all criteria of tumor initiating stem cells, that include expression of stem and progenitor markers like Nestin, Sox-2, Musashi-1, high expression of CD133 (Corti S et al., 2006) and high levels of Aldehyde dehydrogenase (ALDH activity) activity used as an assay system to identify and isolate stem and progenitor cells. Here, we used the CD133 positive, tumor initiating stem cell population of HNGC-2 cells, to evaluate the role of Dlxin-1 on its cell growth and tumorigenecity. Additionally, we used LN18 cells, to compare the effects of Dlxin-1 on cell growth on established glioma cell-lines that are not reported to be containing the CD133 positive BTSCs. The LN18-Dlxin-1 and HNGC-2- Dlxin-1 cells were analyzed for their growth and compared to their parental cells for determining their proliferation potential by MTT assay. As shown in Fig 2A, the HNGC-2 and LN18 cells grew in an exponential manner over the 5 days period. The Dlxin-1 expressing cells exhibited a lowered growth potential exemplifying the role of Dlxin-1 as a growth suppressor. Since, we were interested in studying the effects of Dlxin-1 on brain tumor initiating cells, most of our studies presented here are done with the cancer stem cell-line HNGC-2, unless mentioned otherwise. 103 Next, we analyzed the effect of Dlxin-1 on growth in vitro, by determining the colony formation ability of HNGC-2 cells scored over a 2 weeks period. Both the size and number of colonies were significantly decreased by Dlxin-1 (fig.2 B. a). The EV cells produced 490 ± 11.53 colonies, while the Dlxin-1 cells formed 205 ± 18.02 colonies, indicating a reduction to the extent of 59 ± 4.52 % in colonies number (fig.2 B. b) by Dlxin-1. To further this study on the effect of Dlxin-1 on in vitro transformation, we performed a soft agar assay with both HNGC-2-EV and HNGC-2-Dlxin-1 cells. Expectedly, the Dlxin-1 cells exhibited a decreased clonogenecity in soft agar assay reflected at the level of both colony size (Fig. 2C. a), and colony number quantitatively depicted in (Fig.