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. 2C. b.) a 59.94 % ± 5.08 decrease in clonogenicity was obtained due to
Dlxin-1. This indicated that Dlxin-1 was not functioning as an anti-proliferative protein, but more importantly, inhibiting the transforming potential of these cells. Next, we determined the effect of Dlxin-1 on directional cell motility in vitro was analyzed for
HNGC-2 cells by wound closure migration assay. This Wound healing assay is performed to estimate the migration and proliferation rates of different cells and culture conditions. As shown in Fig. 2D, while the HNGC-2-EV cells within 24 hours were able to repopulate the wound and showed increased cell migration, the HNGC-Dlxin-1 cells, were not able to do so in the same time frame analyzed (Fig. 2 D. a). A 67.05 ± 10.47% decrease in migratory ability was caused by Dlxin-1 compared to EV cells (Fig 2D. b), indicating that Dlxin-1 was effective in suppressing cellular motility in vitro. Glioblastoma being a highly invasive tumor, its progression, frequently leads to infiltration of the tumor cells to the surrounding brain tissues. Hence, it was considered important to analyze if
Dlxin-1 can act as an inhibitor of invasion of glioma stem cells. We have already established the highly invasive nature of the HNGC-2 cells using Matrigel penetration assay. Using similar assay, we found that Dlxin-1 cells exhibited a 66.77 ± 4.91 % lower migration potential compared to HNGC-2-EV ceils (Fig. 2E. a & b) performed over a 22
104 hour interval. Since, localized treatments of GBM are palliative, and there is a lack of success in eradicating this invasive disease, identification and characterization of proteins like Dlxin-1 that can inhibit the invasive behavior of GBM may serve as potential targets for effective therapy.
2.9
2 E i" -•-HHGC2.EV -»-HNWGCH>kln 1 •n 15 1 8... 0 0 12 3 4 5 Days in culture B. a. HNGC2-EV HNGC2-0lxin-1 600
• Ht40c;-rv • Hr«ac2-okti-i
105 D. a. b. HNGC2-EV HNGC2-l)Uin-1 4.5 4 3.6 S c 3 K 2.5 • HMGC3-Et/ o iiHNaC}-0fcin-1 C 2 o n 1.5 u 1 •> 0.5 0 24h 600 E. a. HNGC2-EV HNGC2-0lxin-1 SCO (A « 400 u • moCI-Okb-t I 300 i 200
o 100 o
Fig. 3. 2. Dlxin-1 induces growth inhibition and cell migration in vitro. A. the cell proliferation rate of the parental cells LN18, HNGC-2 and LN18-Dlxin-1 and HNGC-2-Dlxin-1 cells was determined by MTT assay. The OD values were determined at every 24h interval over a period of 5 days. The results of the assay are expressed as Means (±SD) (n=3). B. a. The colony forming Units by Dlxin-1 was determined by transfecting EV and Dlxin-1 cDNA construct into HNGC-2 cells and scoring for G418 ® (700 ug/ml) colonies after 2 weelcs. b). The Colony forming ability of Dlxin-1 on scoring the G418® colonies is shown as CFU/ug of DNA and is quantitatively represented as a histogram (14.78 ±SEM (n=3)). C. a. The HNGC-2-EV and HNGC-2-Dlxin-1 cells were scored for clonogenicity in soft agar after 8 days. The data, is quantitatively expressed in (b) and is obtained by counting colonies from 10 random fields, (2.5275±SEM (n=3)). D. a. Wound closure assays were performed with HNGC-2-EV and HNGC-2-Dlxin-1 cells to analyze the cell migration and proliferation In vitro. The cells immediately after creation of wound (Oh) and after 24h are shown at 10X b). The values represented as migration distance from the site of wound were computed using Image Pro software 6. (0.02416±SEM (n=3)). At 24h, wound closure was complete in HNGC-2 cells, but was considerably slower in HNGC-2 Dlxin-1 cells. E. a. Cell migration assays were performed with HNGC-2 EV and HNGC-2-Dlxin-1 cells. Cells (5x105) were seeded into the top of Transwell migration chambers and allowed to migrate for 22 hours towards medium with serum or media alone serving as negative control placed in the lower chambers. Cells were fixed, and stained using cell stain solution and 10 independent fields were counted and represented quantitatively in b. (30.4835±SEM (n=3))
106 3. 3. Suppression of tumorigenecity with Dlxin-1
To analyze whether, the decrease in proliferation potential and in vitro transformation and migratory capabilities of HNGC-2 Dlxin-1 cells transcended to their effects in vivo analyzed by tumorigenecity assays performed in immuno-compromised mice. We have earlier shown that the HNGC-2 cells in line with their cancer stem cell nature can get implanted intra-cranially with small numbers and establish a tumor there within a week.
Also, the tumor development was very rapid in mice with HNGC-2 cells injected sub- cutaneously, wherein, all the mice (n=3) with HNGC-2 injected cells formed tumors with a tumor volume of 1784.79 mm^± 93.31 within 13 days. The same mice injected with
HNGC-2 Dlxin-1 cells formed small tumors with delayed kinetics displaying a maximum tumor volume of 296.18 mm^± 27.35 SD (Fig 3A,a), translating to a 83.4 ± 1.655 % decrease in tumor volume in all the mice examined (n=3) on the same day (Fig.3A. b) .
While, there are already few reports about MAGED1 being an inhibitor of angiogenesis in endothelial cells (Shen W. G. et al., 2007), it was important to explore its function as an anti-angiogenic protein in the CD133 positive glioma stem cells, infamous for their ability to form highly vascularised tumors. As expected, the tumor tissue analyzed from the HNGC-2 EV cells, expressed CD133 and was highly vascularised as can be seen from high number of blood vessels and strong VEGF positivity as shown in Fig. 3B. The small tumors obtained from Dlxin-1 transfectants, besides being attenuated in tumor size also showed a decrease in number of blood vessels and a decreased VEGF expression
(Fig 2B). Interestingly, the Dlxin-1 cells showed high positivity for CD133, a marker for neural and cancer stem cells (Fig 38).
107 Fig 3. A. a.
HNGC2-Dlxin.l4 • HNOC3-EV CHNGC2-Dbiii-1 HNGC2-EV-4
DAPI VEGF CD133 B.
o z I
Fig. 3. 3. Dlxin-1 exhibits anti-tumorigenic and anti-angiogenic properties in vivo. A. Tumor development and progression was assayed in vivo by implanting 1 x 10' cells of both HNGC-2-EV and Dlxin-1 transfected cells on either side of the flanks of the mouse in NOD-SCID mice. The tumors developed In sHu (shown with an arrow) are shown in (a) and the tumor volumes were determined on d-13 and are quantitatively represented in (b). The data obtained is from one of the representative experiment and is expressed as SEIM±60.33194 (n=3 mice). B. Tumor cells, in tumor tissue sections, of both HNGC-2-EV and HNGC-2-Dlxin-1 derived from their respective xenografts were co-stained with antibodies to proteins VEGF and Cd133 and analyzed at 63X.
108 3. 4. Dlxin-1 lowers the MMP-2 expression in glioma cell-lines
In response to DNA damage, p53 accumulates and functions as a sequence- specific DNA-binding protein, which positively regulates expression of several genes, including p21. Cells then undergo p21-dependent cell cycle arrest, which allows DNA damage repair. To study the role of Dlxin-1 on p21 activation, we analyzed both LN18 and HNGC-2 cells both over-expressing Dlxin-1 for expression of phospho-p21. Western blotting analysis was performed with cell extracts of the empty vector and Dlxin-1 expressing LN18 and HNGC-2 cells and the blots were probed with p21 (total) and p21
(Thr145) antibodies. LN18-Dlxin-1 cells showed an increased p21 expression and a decreased phospho-p21 (Thr145) expression. The HNGC-2 cells did not express p53, and consequently showed no p21 expression, but showed expression of activated p21, that was down-regulated in Dlxin-1 expressing cells (Fig. 4A). Lowering of p-p21 in these ceils is strongly indicative of the role of Dlxin-1 as an anti-proiiferative protein.
This prompted us to look for expression and activation of MMP-2 and MMP-9 proteins that are important in invasion and metastasis for glioma cells. There is a report, that over-expression of NRAGE suppresses metastasis of melanoma and pancreatic cancer probably through down-regulation of MMP-2. Glioblastomas are characterized by an aggressive local growth pattern and a marked degree of invasiveness, resulting in poor prognosis. Tumor progression is facilitated by an increased activity of proteolytic enzymes such as matrix metalloproteinase's (MMPs). Elevated levels of several MMPs were found in glioblastomas compared to low grade astrocytoma and normal brain (NB)
(Chintala et al., 1999; Giese et al., 1996; Forsyth et al., 1999). MMP-2 and MMP-9 are known to be involved in tumor cell invasion, and metastasis. Hence, we undertook to determine the expression and activity of these proteins in both LN18 and HNGC-2 cells.
For this, we used RT-PCR and Gelatin zymography to determine the expression and
109 enzyme activity of MMP-2 in Dlxin-1 over-expressing or Dlxin-1 silenced HNGC-2 and
LN-18 cells. As shown in Fig. 4B, the LN18, HNGC-2 and HNGC-2 tumor cells all showed high levels of MMP-2 expression, visualized as a single 411 bp product by SQ-
RT-PCR using MMP-2 specific primers. A significant decrease in MMP-2 expression was however, noted in all the Dlxin-1 over-expressing cells. Quantitatively, the expression levels were down by 3.3 fold for LN18 cells, 1.8 fold for HNGC-2 cells with a marked lowering by about 6.25 fold in tumor derived HNGC-2 cells (Fig. 48,a). Expression of beta-actin served as a loading control. These expression studies very well corroborated with the MMP-2 activity assays performed by Gelatin zymography. Here, we found that
Dlxin-1 over-expressing LN18 and HNGC-2 cells both, showed a lowered MMP-2 enzyme activity (Fig. 48, b). RNA interference with Dlxin-1, increased expression of
MMP2 in LN18 by 179% and by 137% in HNGC-2 cells (Fig.4C), thereby attributing the lowered invasiveness assayed as MMP-2 activity, to presence of Dlxin-1. Using gelatin zymography, we found a decrease in MMP-9 activity for HNGC-2 cells (Fig.48, b), while no MMP-9 activity was evident in LN18 cells. These studies elaborate, that Dlxin-1 can induce a down-regulation of both MMP-2 and MMP-9 in cancer stem cells and thereby contribute to inhibition of glioma invasion.
110 Fig. 3. 4. Dlxin-1 suppresses expression ana activiTy of MMP2 and IVIMP-9. A. Glioma cells LN18, cancer stem cells HNGC-2 and their corresponding cell-lines over-expressing Dlxin-1 were serum starved overnight and then stimulated with medium containing serum for 30 minutes and analyzed for expression of cell cycle regulatory proteins p21 and phospo-p21 (Thr145) by Western blotting. B. a. The expression of MMP-2 was assayed by RT-PCR using gene specific primers for the indicated cell-lines. B. b. The effect of Dlxin-1 on invasion was studied by analyzing the activity of IMMPs, IMMP-2 and MIMP-9 by performing gelatin zymography on both LN18 and l-INGC-2 cells and their respective stable transfectants. C. The expression of IWMP-2 was assayed by RT-PCR using gene specific primers for the indicated cell-lines. In separate experiments, each of LN18 and HNGC-2 cells were transiently transfected with respective Dlxin-1 siRNAs and then analyzed for MIMP-2 activity. Each of these cells transfected with the transfection mix without the Dlxin-1 siRNA, served as a IMock control, p- actin served as a loading control.
Ill 3. 5. Down-regulation of proliferation associated genes by Dlxin-1
The inhibitory effects of Dlxin-1 on cell proliferation, tumorigenecity and invasion prompted us to acquire a comprehensive global expression profile of cells wherein, we compared the differential expression of genes, between HNGC-2 and Dlxin-1-HNGC-2 cells (Fig 5. A.). A detailed comparative analyses of the changes in expression levels between the 2 cell types showed that Dlxin-1 induced significant changes in the expression of genes associated with growth regulation of 66 signature genes involved with proliferation, growth inhibition from global gene expression. This is exemplified with the help of cluster analyses of these genes represented in Fig. 5. B. a. and is enlisted in
Table 3. 1. A Scatter plot for these representative genes is shown in Fig. 58. b.. A marked up-regulation of genes involved with tumor suppressor functions like VHL, p53AIP1 and GADD45B and down-regulation of genes involved with proliferation like
Egr-1 and metastasis associated genes like p311 were seen to be manifested by Dlxin-
1, reaffirming its role Dlxin-1 as an anti-proliferative protein.
Up regulated Tumor suppressor: RAD54L, (6.4)VHL(1.98) , TP73 (1.94), GLTSCR1 (1.9875), ST14 (1.82) , FLJ12438 (1.64), PANX2(1.879), PANX3 (1.749) Differentiation: GDF1 (2.76), BM88 (2.476) , TUBB4 (1.788) , DRD2 (2.66) , DRD4 (1.972786) ,NGFB (1.86048) , AD7C-NTP(2.710894) Apoptosis UNC5A(2.811606), PDCD1 (2.200467), GADD45B(19.8489), PLEKHF1 (2.060324), PAK6(1.807517), P53AIP1 (3.262119) Axonogenesis; cell BAH (1.839706) migration; roliferation: drug-sensitive proteini (8.621387) IVIethylation: HIC2(2.236821) Down regulated Oncogenes: PP311(2.690119), PVRL3(1.851678), RAP2B(2.023039), RAP2C(2.960041), RASA2(1.533375), RAB10(1.528773), KRAS(2.03579) Transcription E2F3(3.630556), ELF2(1.665517), EGR1 (8.721383), TCEB3(2.00235), regulators: EIF2B3(3.247609), EIF3S1 (1.590516), EIF3S10(2.172478), EIF4ENIF1 (3.509265) MAP kinase: MAP3K11 (1.979556),MAP4K4(1.705614), MKNK1 (3.398181)
Table 3. 1. Micro array Expression analysis of signature genes associated with proliferation, growth inhibition and Transcription were compared with HNGC-2-Dlxin-1 cells to HNGC-2-EV. Values are change in fold value.
112 j>**»t-jaip
L wnnOTCvnv L4CW4 n*»a:. nxiw ^»>(< ».Ti. £-.ii>L<*»c*.n«r>-*'je» i«('^<-urtHiEc>^*> [w*v—t •^:f. K<«y C Iter ru--avt* r t*^ • {i(na(W«P»IU.ILA > •'•r rrrrriniFMaiiw • HNGC-2-DlxJn-1 HNGC-2-EV 13 B. b. HNGC-2-EV HNGC-2-Dlxin-1 «-n— aoi Flg^^^Dbdn-^uppresse^h^xpreMlor^^ene^nvolw^r^^ transcription level. A. Global transciptome analyses was performed witli RNA from HNGC-2- EV (p64) and HNGC-2-Dlxin-1 (Clone 8) cells and hybridized to Agilent- oligo arrays. The Tree map of the differentially expressed genes tietween HNGC-2 and K1NGC-2-Dlxin-1 cells is shown. B. a. The Tree map of differentially expressed genes, specifically involved in proliferation, between HNGC-2-EV (p64) and HNGC-2-Dlxin-1 (Clone 8) cells. Genes that were 2 fold and above differentially regulated were clustered using Gene Spring GX v 7.3.1 software and Excel, using gene tree algorithm under standard correlation matrix. Color bar indicates the gene expression profile. Red indicates higher expression and Green indicates lower expression. B. b. Gene expression patterns of few of the genes important in cell proliferation function chosen from the Tree map is represented as a Scatter plot. Blue lines indicate two fold changes in gene expression levels between the paired cell types. Genes over-expressed in HNGC-2-EV cells (p65) compared with HNGC-2-Dlxin-1 (clone 8) are shown in red; those under-expressed are shown in yellow. Positions of few of the important genes in scatter plots are Indicated in black. The gene expression levels for the data shown in A and B was normalized with Gene Spring GX and Excel, using the recommended Per Chip and Per Gene Data Transformation: Set measurements less than 0.01 to 0.01, Per Chip, Normalized to 50th percentile Per Gene, Normalized to Median. 114 3. 6. P311 identified as a target for Dlxin-1 In an attempt to investigate, the targets with which Dlxin-1 could possibly interact in vivo to manifest its biological function, we analyzed the interactions of different proteins with Dlxin-1 (Koike A et al., 2003; 2005). Using an advanced version of the "Kinase Pathway Database", known as PRIME we came across a new branch of Dlxin-1 interacting proteins. These newly found proteins are P311, Frat 2, Rad and Cacngl. Amongst, the various interacting proteins identified, we focused on the protein P311, due to its known role in glioma invasion and metastasis. The Dlxin-1-HNGC-2 cells showed a lower expression of P311 compared to HNGC-2-EV cells in micro-array analyses. We validated this data with Q- RT PCR and found that with Dlxin-1, there was a 80% ± 0.0404 SD decrease in P311 expression. This signifies the role of P311 as a putative interacting partner for Dlxin-1 in exhibiting the tumor suppressor function in HNGC-2 cells. Fig. 6. A. B. s • HNGC2^V £ 0.6 DHNGC2-Db(in- c Z 0.4 « 0.2 O 0 Targets prediction for Dlxin-1 Fig. 3. 6. Down-regulation of P311. A. An Interaction tree predicting potential Dlxin-1 targets generated using PRIiME database. The thicit blue lines show known targets and the thin blue lines show the predicted potential targets. B. Expression of P311 by q-PCR analyses in HNGC-2-EV and HNGC-2-Dixin-1 cells. The data is a representative of 3 independent experiments and Is expressed as fold change with respect to HNGC2-EV after normalization with beta-actin and is represented as SD ± 0.040418 115 3. 7. The cells over-expressing Dlxin-1 exhibit increased attributes of sternness We in our earlier studies have provided evidences that establish the cancer stem cell nature of HNGC-2 cells. These cells arose by spontaneous transformation of non- tumorigenic neural stem cells HNGC-1. The HNGC-2 Dlxin-1 cells were analyzed using cDNA micro-arrays to reveal their molecular signatures and compared to their un- transfected counterparts. We found a spectacular increase in expression of genes important in establishment and maintenance of stemness, induced due to Dlxin-1 in HNGC-2 cells. Appreciably, a total of 144 signature genes associated with stemness were found to be highly over-expressed in HNGC-2-Dlxin-1 cells. Amongst, these genes, few genes are well reported and known to be important for specification of embryonic stem cell fate like those belonging to the well-characterized ALDH, Sox and Klf families (Table 3. 2). The cluster analysis performed for the selected genes based on their functional annotation as stemness genes is depicted in tree diagram (Fig. 7A). The scatter plot shown in (Fig. 7. B) additionally depicts the increased stemness signature in these cells (Fig. 7B). An overall increase, at least to an extent greater than 2 fold is manifested for genes like ALDH and sox proteins that are important in establishment and maintenance of stem cell fate. In addition, the LIM domain finger proteins and various important transporter proteins were up-regulated by Dlxin-1 (Table 3. 2). 16 Gene Symbo 1 fold change( Product Up Regulate(i ALDH1B1 2.27633303 aldehyde dehydrogenase 181 precursor ALDH3B2 2.21115049 aldehyde dehydrogenase 3B2 ALDOA 2.79773541 aldolase A ATXN7 2.802937322 ataxin 7 ATXN7L4 1.740281924 ataxin 7-like 4 CREB3L3 2.3029355 cAMP responsive element binding protein 3-like 3 CHRDL1 2.418779698 chordin-like 1 MIST1 1.875737411 class II bHLH protein MIST1 CNIH2 4.21638813 cornichon homolog 2 C0R06 2.921979628 coronin 6 DVL3 1.707200005 dishevelled 3 EFHC2 2.577255914 EF-hand domain (C-terminal) containing 2 EN2 2.521127138 engrailed homolog 2 ESPL1 3.33673076 extra spindle poles like 1 FBX011 1.843075036 F-box only protein 11 isoform 3 FLJ14768 1.969465847 FLT3-interacting zinc finger 1 F0XA3 1.869718545 forkhead box A3 F0XB1 1.671700793 forkhead box B1 FOXC1 1.794221509 forkhead box CI FOXE1 1.510155524 forkhead box El FOXH1 1.251954894 forkhead box HI FOX03A 2.541443176 forkhead box 03A F0X03A 2.352162021 forkhead box 03A FZD9 1.787747223 frizzled 9 FLJ22688 3.155946735 fuzzy homolog FYB 1.83226827 FYN binding protein (FYB-120/130) isoform 1 FYB 2.468364135 FYN binding protein (FYB-120/130) isoform 1 FZR1 1.695875569 Fzrl protein GABPB2 4.020359231 GA binding protein TF, beta subunit 2 isoform gamma 1 GLIS2 2.120955576 GLIS family zinc finger 2 HIST1H2AE 1.527550024 H2A histone family, member A HIST1H2AJ 3.075336119 H2A histone family, member E AFZ 2.186746736 H2A histone family, member Z HIST1H2BD 2.222544996 H2B histone family, member B HIST1H2BN 2.123154747 H2B histone family, member D HIST1H2BM 2.060685312 H2B histone family, member E HIST1H2BB 2.264286455 H2B histone family, member F HIST1H2BF 2.124133677 H2B histone family, member G HIST1H2BE 2.279335422 H2B histone family, member H HIST1H2BH 2.140853878 H2B histone family, member J HIST1H2BI 2.258930756 H2B histone family, member K HIST1H2BJ 2.278739338 H2B histone family, member R H2BFS 2.238775831 H2B histone family, member S HIST1H3B 1.974461061 H3 histone family, member L HIST3H2BB 2.140456021 histone H2B INA 1.535597783 internexin neuronal intermediate filament protein, alpha JUN 3.680672204 jun oncogene FLJ38753 1.480151317 kelch domain containing 7A KLHL14 1.960630145 kelch-like 14 KLHL6 2.479175114 kelch-like 6 KLF2 1.340198572 Kruppel-like factor KLF13 1.03243688 Kruppel-like factor 13 KLF14 2.434245685 Kruppel-like factor 4 (gut) KLF5 1.219091263 Kruppel-like factor 5 KLF6 1.277534356 Kruppel-like factor 6 KLF6 1.305666325 Kruppel-like factor 6 KLF7 3.894532862 Kruppel-like factor 7 (ubiquitous) 117 FLJ22386 3.801280963 leucine zipper domain protein LHX1 1.578784991 LIM homeobox protein 1 LMX1A 1.358779186 LIM homeobox transcription factor 1, alpha isoform c LMX1B 1.454995259 LIM homeobox transcription factor 1, beta MAMDC2 5.201646546 MAM domain containing 2 MXD3 3.335167747 MAX dimerization protein 3 MS4A1 4.498783111 membrane-spanning 4-domains, subfamily A, member 1 KIAA1193 2.084114311 mesoderm induction early response 1, family member 2 MSI1 1.77936137 musashi 1 NES 1.185120325 nestin NOG 1.637283467 noggin precursor NOTCH4 1.820525365 notch4 preproprotein UNC5A 2.811606275 netrin receptor Unc5h1 NPAS3 1.887204718 neuronal PAS domain protein 3 isoform 1 AD7C-NTP 2.710894221 neuronal thread protein AD7c-NTP NKX2-5 1.459606297 NK2 transcription factor related, locus 5 NYX 1.978512008 nyctalopin 0LFML3 3.505341363 olfactomedin-like 3 OR10A4 1.876587118 olfactory receptor, family 10, subfamily A, member 4 0R11A1 1.837046155 olfactory receptor, family 11, subfamily A, member 1 0R2H1 2.15724351 olfactory receptor, family 2, subfamily H, member 1 0R5A1 2.387194133 olfactory receptor, family 5, subfamily A, member 1 OR8B8 2.522602696 olfactory receptor, family 8, subfamily B, member 8 0R8H1 1.728031678 olfactory receptor, family 8, subfamily H, member 1 PAX3 3.002565461 paired box gene 3 isoform PAX3a PAX4 2.52504815 paired box gene 4 PTCH2 1.96310504 patched 2 PDLIM2 2.139099365 PDZ and LIM domain 2 isoform 2 PDLIM5 2.398309304 PDZ and LIM domain 5 Isoform b PDZK4 1.620081136 PDZ domain containing 4 PDZK7 2.84432229 PDZ domain containing 7 PHF15 3.289658677 PHD finger protein 15 PHF15 3.25553192 PHD finger protein 15 PHF16 1.860262088 PHD finger protein 16 PHF19 2.466339796 PHD finger protein 19 isoform b PHF20 2.124435179 PHD finger protein 20 PCGF1 1.139349709 polycomb group ring finger 1 POU3F4 1.717769424 POU domain, class 3, transcription factor 4 POU6F2 1.825034022 POD domain, class 6, transcription factor 2 REN 1.87193029 renin precursor RC0R3 1.540930147 REST corepressor 3 RKHD1 1.844064398 ring finger and KH domain containing 1 RKHD1 1.718075477 ring finger and KH domain containing 1 RNF121 1.981807404 ring finger protein 121 isoform 1 RNF38 2.500635907 ring finger protein 38 isoform 1 RBM15 2.723453054 RNA binding motif protein 15 RUSC2 2.546349488 RUN and SH3 domain containing 2 APS 2.924234386 SH2B adaptor protein 2 SPN 1.792930284 sialophorin SMO 1.385144832 smoothened ARGBP2 1.656843546 sorbin and SH3 domain containing 2 isoform 1 SNX26 1.872473023 sorting nexin 26 SNX26 1.955445536 sorting nexin 26 FLJ14800 1.888364787 SPRY domain containing 3 SSB1 3.475771906 SPRY domain-containing SOCS box protein SSB-1 SSB3 2.518011933 SPRY domain-containing SOCS box protein SSB-3 SSB3 1.400019831 SPRY domain-containing SOCS box protein SSB-3 SSB3 1.716517426 SPRY domain-containing SOCS box protein SSB-3 _SSB4 2.05302960.'; 118 S0X1 1.342438714 SRY ^ex determining region Y)-box 1 SOX2 2.01153586 SRY {sex-determining region Y)-box 2 S0X3 1.446597993 SRY (sex determining region Y)-box 3 S0X8 1.992196539 SRY (sex determining region Y)-box 8 S0X17 1.875822567 SRY-box 17 SOX18 1.316923631 SRY-box 18 SOX21 2.049486599 SRY-box 21 SOX7 2.114550518 SRY-box 7 STIVIN1 3.906016116 stathmin 1 SUSD3 2.386039574 sushi domain containing 3 SYT3 1.599663455 synaptotagmin 3 SNPH 1.568538854 syntaphilin TFAP2E 2.063453418 TF AP-2 epsiion (activating enhancer binding protein 2 epsiion) MAFF 3.623409289 transcription factor IVIAFF TUBB 1.771841718 tubulin, beta polypeptide UTF1 1.962246419 undifferentiated embryonic cell transcription factor 1 WNT3A 2.069407845 wingless-type MMTV integration site family, member 3A ZNF223 2.197210245 zinc finger protein 223 Down regulated KIFC2 7.913652428 Idnesin family member C2 VTI1A 2.979530487 SNARE Vtila-beta protein isoform b CLDN12 2.348182523 claudin 12 MAT2A 5.787860874 methionine adenosyltransferase II, alpha IVIAT2A 7.068036152 methionine adenosyltransferase II, alpha PCTK2 2.773348569 PCTAIRE protein kinase 2 PSEN1 1.478994729 presenilin 1 Table 3. 2. Micro array Expression analysis of signature genes associated with sternness was compared with HNGC-2-Dlxin-1 cells to HNGC-2-EV. Values are change in fold value. 119 Fig. 3. 7. A HMOC-2-CM)iin-1 HNGC-2-EV 120 B. lOOCh HNGC-2-EV Fig. 3. 7. Identification of stemness Genes up regulated by Dlxin-1 in HNGC-2. A. The Tree map of the different expressed genes between HNGC-2 and HNGC-2-Dlxin-1 cells important in stemness function. Genes that were 2 fold and above differentially regulated were clustered using Gene Spring GX v 7.3.1 software and Excel using gene tree algorithm under standard correlation matrix. Color bar indicates the gene expression profile. Red indicates higher expression and Green indicates lower expression. B. Gene expression patterns of few of the genes important in stemness function chosen from the Tree map, highlighting the differential levels in gene expression is represented as a Scatter plot. Blue lines indicate two-fold changes in gene expression levels between the paired cell types. Genes over- expressed in HNGC-2-Dlxin-1 cells (Clone 8) compared with IHNGC-2-EV (p65) are shown in green and the positions of few of the genes important in stemness function in scatter plots are marked in black. The gene expression levels for the data shown in C and D was normalized using Gene Spring GX and Excel, using the recommended Per Chip and Per Gene Data Transformation: Set measurements less than 0.01 to 0.01, Per Chip, Normalized to 50th percentile Per Gene, Normalized to IMedian 121 Fig. 3. 8. Genes important in Sternness function are significantly up-regulated by Dlxin-1 in HNGC-2 cells in comparison to HNGC-2-EV cells We validated microarray data by using multiple approaches that included real time analyses for expression of genes important as stem cell markers such as Sox 2, cMyc, ABCG2, Klf4 and OctSM by Q- RT PCR (Fig. 8A) and further validation by Immune fluorescence (Fig. 8B) and Western blotting (Fig 8C). This increase in the stemness signature induced by Dlxin-1 and exemplified in global transciptome analyses \Nas also evident in functional assays like the Neurosphere generation assay, used to evaluate stemness. The HNGC-2 Dlxin-1 cells showed an increased ability to form Neurospheres. This increased ability was apparent in terms of an increase in the number and size of the developed Neurospheres is shown in (Fig.SD. a) and is quantitatively represented in (Fig. 8D. b). 122 A. a. • KLM nellYC •Aecoi • (0»U DOCTM _ i HNGC 2 EV HNGC 2 DIxin 1 DAPI Antibody Merged DAPI Antibody Mef^d 1 1 CD133 S0X2 1 1 SOX10 KLF4 ABCQ2 cMYC 1 1 Da Neurospti«re assay • HNGC2-EV 1 1 pcMYC 1 1 GFAP 1 1 NMtIn Fig. 3. 8. Genes important in Stemness function are significantly up-regulated by Dlxin-1 in HNGC-2 cells in comparison to HNGC-2-EV cells. A. Real-time PCR analysis of gene expression in Dlxin-1 HNGC-2 transfectants to vector control cells. The data is a representative of 3 independent experiments and is expressed as fold change with respect to HNGC2-EV after normalization with beta-actin and is represented as SD±ABCG2, cMyc, Klf4, Sox2 and Oct3/4 - 1.679216, 0.396036, 0.193537, 11.06168276, 2.401709806. B. Immuno-fluorescence analyses of HNGC-2 EV and HNGC-2- Dlxin-1 cells using antibodies specific to stemness mariners SOX-2, SOX-10, CD133, KLF4, cMyc, pcMyc, GFAP,ABCG-2, Nestin (red). Nuclei were stained with DAPI (blue). Scale -10 um. C. immuno-blot analysis using cell lysates of HNGC-2 EV and HNGC-2- Dlxin-1 cells for KLF4, SOX-2, SOX-10 and ABCG-2 proteins. The same blots were stripped and stained with beta-actin serving as a loading control. D. (a) HNGC-2-EV and HNGC-2-Dlxin-1 cells form free-floating Neurospheres, within 7days, when grown in NBM containing EGF (20 ng/ml) and bFGF (20 ng/ml). HNGC-2-Dlxin-1 cells form larger and higher number of Neurospheres (10X). The Neurospheres formed on D10 by each of the cell-line were counted in 10 random fields and the data is quantitatively represented in (b) and the values plotted are (SEM ± 4.814989 (n=3)). ' "^ 123 Discussion Gliomas bear a dismal prognosis, show high aggressiveness, and display propensity for recurrence. Recent, studies have identified a small subpopulation of tumor initiating cells or cancer stem cells from Gliomas (Singh SK et al., 2003), which distinctly possess the ability to self-renew (Hemmati HD et al., 2003), initiate the tumor with very small cell numbers, drive tumor progression (Singh SK et al., 2004) and contribute to therapeutic resistance due to the expression of several ABC transporters (Rich JN et al., 2007). Hence, it would be imperative to develop therapeutic strategies to target genes specifically involved in stem cell self-renewal and proliferation and in the cancer stem signaling pathways (Ward RJ et al., 2007). We have reported the development and characterization of a bonafide tumor stem cell-line HNGC-2 from a glioma tissue (Shiras et al., 2003). The cells are enriched with high number of CD133 positive glioma stem cells. In this study, we report on the role of Dlxin-1 acting as an anti-proliferative protein for glioma cell-lines like LN18 and more importantly, for the Cd133 enriched glioma stem cells HNGC-2, thereby, highlighting its relevance as an effective target gene for cancer stem cells. The HNGC-2- cells exhibited a dramatic decrease in proliferation, clonogenicity, tumor grovi^h and in vitro invasive potential with Dlxin-1 in comparison to the parental HNGC-2 cells. Expression data using whole genome oligo-nucleotide Microarrays similarly reveal a noteworthy inhibition by Dlxin-1 of a whole array of genes associated with proliferation (Fig. 3. 5. b). A similar growth inhibition was effected in LN18 cells, but the effects were manifested at the in vitro levels as these cells failed to form tumors under similar experimental conditions to the ones used for HNGC-2. Importantly, the HNGC-2 cells could form large tumors intra-cranially with as few as 100 cells (Shiras et al., 2007). These findings are in line with the cancer stem cell hypothesis which states that only a small number of CD133 positive cancer stem cells are capable of re- 124 capitulating the original tumor in vivo (Singh SK et al., 2004). Though, there are contrasting reports about the selectivity of tumor formation with only CD133 positive cells there are now some recent studies which suggest that the tumor initiating potential in vivo also resides with the CD133 negative cells (Joo KM et al., 2008). This may possibly be due to the heterogeneity existing in GBM wherein the tumor mass may contain cells of different types with varied tumor initiation potential. Experimental evidence indicates that resistance of these tumors to current therapies is attributable to a small subpopulation of fast-growing cancer stem cells (Liu G et al., 2006). Our studies have shown that HNGC-2 cells were highly radio and chemo-resistant (unpublished data). Hence, molecular therapeutic strategies designed to specifically target this tumor initiating population would have an immense potential in management of tumors like GBM that are refractory to conventional treatment regime. The efficacy with which Dixin- 1 was able to inhibit HNGC-2 glioma stem cells in vitro as well as in vivo was encouraging. There are already reports that Dlxin-1 is inhibitory on pancreatic and melanoma cell-lines wherein the growth suppression is manifested through MMP-2 activity (Chu, C.S. et al., 2007). With both LN18 and HNGC-2 glioma cells, we did find significant inhibition in MMP-2 expression and activity. A significant increase of nuclear p21 and a decrease in cytoplasmic p21 accumulation seen with Dlxin-1 in LN18 cells is indicative of the activation of checkpoint proteins with Dlxin-1. We could identify several other novel targets of Dlxin-1 that may be contributing to the anti-proliferative responses of Dlxin-1 and have not been reported earlier. We predicted P311 to be a functional target of Dlxin-1, using the database PRIME (Koike A et al., 2003; 2005). This predicted protein was validated to be a functional target by us, since, we find that P311 levels were significantly down-regulated (> 4 fold) in Dlxin-1-HNGC-2 cells, which are attenuated in their tumorigenic and migratory abilities. P311 is a 8 KDa neuronal protein highly expressed in invasive glioma cells and is important in regulating glioma motility and 125 invasion through the reorganization of actin cytoskeleton at the cell periphery (Mariani L et al., 2001; McDonough et al., 2005). Hence its interaction prediction with Dlxin-1 is significant for further deciphering the function of Dlxin-1 as a suppressor of cell migration (Salehi et al., 2000; Wen et a!., 2004; Tian et a!., 2005; Shen et al., 2007). A further detailed analysis is required to exemplify the role of Dlxin-P311 interaction in the process of tumor progression. The other predicted targets are Cacngi, Frat2 and Rad as shown in Fig.8. Additionally, we find an increase in a large number of calcium channel proteins in our Microarray experiments indicating the possibility of a functional interaction of Dlxin-1 with these proteins. Besides the effect on Dlxin-1 on inhibition of proliferation, we observe a persistent increase in expression of molecules involved in stemness with Dlxin-1. The molecules highly up-regulated are genes involved in pluripotency like klf-4, sox-2, c- cMyc and neural progenitor markers like Nestin and Cd133. The microarray data indicated a similar trend wherein multiple members of the SRY and Klf family of proteins were up-regulated. The finding that Dlxin-1 decreased the growth of brain tumor stem cells and yet the cells showed increased features of stemness requires further study. It is possible that with Dlxin-1, the BTSCs are getting attenuated in their proliferative responses and getting converted from BTSCs to stem and progenitor cells, leading to enhanced features of stemness. In conclusion, we demonstrate that Dlxin-1 possesses anti-proliferative activity on glioma cells and more on the chemo and radio-resistant CD133 enriched glioma stem cell population. Inhibition by Dlxin-1 was manifested in terms of suppression in cell proliferation, tumor initiation, progression and invasion in the glioma stem cells. Besides, decreased tumorigenecity, there was an increase in checkpoint protein responses as seen by an increased p21 and decreased angiogenesis in the CD133 positive cells, assayed specifically on the basis of Immuno-reactivity to VEGF. GDI33 positive Dlxin-1 126 cells showed increased ability to form Neurospheres and displayed a global increase in stemness associated genes. Interestingly, the genes associated with pluripotency, like the members of the Klf, sox and LIM domain family proteins were significantly up- regulated. This plausibly suggests the role of Dlxin-1 in inducing reprogramming of the genome followed by conversion of brain tumor stem cells to embryo-like stem or progenitor population that is highly pluripotent. We propose that Dlxin-1 is a valid therapeutic target that can act and inhibit the glioma stem cell population, and thereby lead in effecting better therapeutic management for gliomas. 127 Conclusions • The MAGE proteins are localized on the X-chromosome, Members of this family are classified into 2 subgroups- Subgroups I and II. Members of Subgroup I named as cancer/testis (CT) antigens, comprise of MAGE-A, B, and C and consist of antigens whose expression is generally restricted to tumor cells, or germ ceils, but are not seen in other normal tissues. Proteins and peptides derived from some of these genes have been used in clinical trials for cancer immunotherapy's. In contrast, subgroup II MAGE proteins are expressed in various normal adult human tissues and include proteins of the MAGE D family. Yet, the normal physiological role of MAGE proteins has remained a mystery. • Dlxin-1 a mouse homolog of MAGE-D1, encodes a 775-aa protein that has a partial homology with Necdin at the C terminus, 25 repeats of hexapeptides (WQXPXX) in the middle region and the predicted molecular mass of the protein is 85.7 kDa. • Dlxin-1 3.2 Kb transcript was detected only in sarcomas and neuro-ectodermal tumors. Characteristically, lymphomas, leukemia's, adeno-carcinomas, and carcinomas did not express Dlxin-1. • Dlxin-1, cDNA (2.64 Kb) is cloned from mouse embryo into a mammalian expression vector pTargetT. Stable transfects of Dlxin-1 over expressing NIH3T3-Dlxin-1, PC12- Dlxin-1, LN18-Dlxin-1 and HNGC-2-Dlxin-1 clones were generated. • Dlxin-1 over expression in neuronal cell system like PCI2 cells shows accelerated differentiation and neurite outgrowth in response to nerve growth factor (NGF). The differentiation was manifested in as early as 24 hours of NGF treatment. A very rapid, marked and sustained up-regulation of MEK and ERK activities were induced by Dlxin-1. Dlxin-1 over expression in PCI2 cells led to simultaneous activation of She 128 and Src pathways. Also an early activation of Akt 308 and Akt 473 was seen in Dlxin- 1 in PC12 cells. • Through our study it has been substantiated that MAP Kinase pathway is activated by binding of Dlxin-1 to NGF receptors p75NTR and Trk A, thus freeing Trk A from inhibitory constraint imposed by p75NTR. • Our studies show that the synergistic interactions of Dlxin-1 with TrkA and p75NTR led to early and enhanced activation of ERK and Akt pathways which play a pivotal role in the phenomena of accelerated differentiation in the PC12 neuronal cell system. • Significant up-regulation of a neuron-specific growth suppressor viz, Necdin, is seen in Dlxin-1 over-expressing PC12 cells. • Dlxin-1 is weakly expressed in cancer stem cells HNGC-2, compared to other established glioma cell-lines. Over-expression of Dlxin-1 effectively inhibits proliferation, tumorigenecity and invasion in HNGC-2 cells by down regulation of MMP2 and MMP9. • GDI33 positive HNGC-2-Dlxin-1 cells showed increased ability to form Neurospheres and displayed a global increase in stemness associated genes. • We proposed, a dual role for Dlxin-1, one as an anti-tumorigenic protein and second as a stemness inducer that converts the highly malignant cancer stem cells to cells that resemble neural stem cells • Since, localized treatments of GBM are palliative, and there is a lack of success in eradicating this invasive disease, identification and characterization of proteins like Dlxin-1 that can inhibit the invasive behavior of GBM may serve as potential targets for effective therapy. 129 Future Studies In order to fully characterize the structure-function analysis of Dlxin-1, the growth suppression, differentiation and Stemness aspects of Dlxin-1 function need to be investigated further. In this study we showed Dlxin-1 binds to both p75NTR and TrkA however, little has been found as to the downstream activities of Dlxin-1 after p75NTR and Trk A interaction. Future studies must address this issue. Necdin maintains the post-mitotic state of neurons. The presence of Necdin seen only in Dlxin-1 transfectants of PC 12 cells and its co-localization with Dlxin-1 in the over- expressing cells makes us speculate an important role of Dlxin-1 in inducing cell cycle arrest, leading to neuronal survival and enhanced differentiation seen in these cells. Is there any direct interaction between Dlxin-1 and Necdin has to be studied using pull down assays. We predicted P311 to be a functional target of Dlxin-1, using the database PRIME. P311 is an 8 kDa neuronal protein highly expressed in invasive glioma cells and is important in regulating glioma motility and invasion through the reorganization of actin cytoskeleton at the cell periphery. A further detailed analysis is required to exemplify the role of Dlxin-P311 interaction in the process of tumor progression. Our results indicate Dlxin-1 decreases the growth of brain tumor stem cells and simultaneously the cells show increased features of sternness.. It is possible that with Dlxin-1, the BTSCs are getting attenuated in their proliferative responses and getting converted from BTSCs to stem and progenitor cells, leading to enhanced features of stemness. Future studies must address this issue. 130 Publications E. Maheswara Reddy, Varsha sepal, Nagesh C Shanbhag, Shiras A. Dlxin-1, a MAGE family protein, induces enhanced neuronal differentiation in PC12 cells through early activation of MEK and Akt pathways. (In communication) E. Maheswara Reddy, Chettiar ST, Nagesh C Shanbhag, Rajendran G, Bakle Anand A, Shiras A. Dlxin-1, a member of the MAGE-D1 family, inhibits cell proliferation yet increases stemness in glioma stem cells. (In communication) Sheetal Dhar, E. Maheswara Reddy, Anjali Shiras, Varsha Pokharkar & B L V Prasad; Natural Gum Reduced / Stabilized Gold Nanoparticles: A Generic System for Drug Delivery Formulations. (Chemistry—A European Journal, 10th Oct, 2008) 131