Abstract
Focal adhesion is known to be highly expressed and activated in glioma cells. Recently, we demonstrated that FAK autophosphorylation inhibitor, Y15 significantly decreased tumor growth of DBTRG and U87 cells, especially in combination with temozolomide. In the present report, we performed gene expression analysis in these cells to reveal genes affected by Y15, temozolomide and combination of Y15 and temozolomide. We tested the effect of Y15 on gene expression by Illumina Human HT12v4 microarray assay and detected 8087 and 6555 genes, which were significantly either up- or down-regulated by Y15-treatment in DBTRG and U87 cells, respectively (p<0.05). Moreover, DBTRG and U87 cells treated with Y15 changed expression of 1332 and 462 genes more than 1.5 fold, p<0.05, respectively and had 237 common genes affected by Y15. The common genes up-regulated by Y15 included GADD45A, HSPA6 (heat-shock 70); DUSP1, DUSP 5 (dual-phosphatase 5); CDKN1A (p21) and common down-regulated genes included kinesins, such as KIF11, 14, 20A, 20B; topoisomerase II, TOP2A; cyclin F; cell cycle protein: BUB1; PARP1, POLA1. In addition, we detected genes affected by temozolomide and by combination of Y15 and temozolomide treatment in U87 cells. Among genes up-regulated by Y15 and temozolomide more significantly than by each agent alone were: COX7B; interferon, gamma-inducible transcript: IFI16; DDIT4; GADD45G and down-regulated: KIF3A, AKT1; ABL; JAK1, GLI3 and ALDH1A3. Thus, microarray gene expression analysis can be effective in establishing genes affected in response to FAK inhibitor alone and in response to combination of Y15 with temozolomide that is important for glioblastoma therapy.
Keywords: Focal Adhesion Kinase, Y397 site, autophosphorylation, brain, glioblastoma, inhibitor
INTRODUCTION
Glioblastoma is the most malignant form of human gliomas and is characterized by high invasion of the normal brain tissue and high resistance to chemotherapy [1]. Thus, it is important to develop novel therapies to inhibit glioblastoma tumor growth and invasion.
Focal Adhesion Kinase (FAK) was shown to be overexpressed in many types of tumors, including brain [2]. FAK plays significant roles in survival, adhesion, invasion, proliferation, and angiogenesis. FAK mediates signaling from the extracellular matrix to the cytoplasm and nucleus and regulates many intracellular processes.
Brain tumors not only overexpressed FAK but also overexpressed autophosphorylated (activated) FAK [2]. Since FAK is highly autophosphorylated in glioblastoma tumors, we tested the FAK autophosphorylation small molecule inhibitor of FAK, called Y15 or inhibitor 14 to block glioblastoma tumor growth [3–5]. We demonstrated that Y15 decreased viability, clonogenicity and tumor growth in two glioblastoma cell lines: DBTRG and U87, especially in combination with temozolomide [6]. In this report, we performed Illumina microarray analysis of Y15-treated DBTRG and U87 cells to test the down-stream signaling mediated by Y15. We found that 8087 and 6555 genes were affected by Y15 in both cell lines with p<0.05. There were 1332 and 462 genes that were affected by Y15 more than 1.5 fold, p<0.05 in DBTRG and U87 cells, respectively and among these genes there were 237 common genes that play role in survival, motility and cell cycle. Thus, this is the first report that demonstrates genes affected by FAK autophosphorylation inhibitor Y15 and by its combination with temozolomide in glioblastoma cells that can be important for future therapy.
MATERIALS AND METHODS
Cell Lines
The early passages of patient-derived human DBTRG glioblastoma cells were obtained Dr. Brian Eliceiri (a kind gift from Dr. Kruse [7]) and maintained in Dulbecco's modified Eagle's minimum essential medium supplemented with 10% fetal bovine serum with 1 µg/ml streptomycin, µg/ml L-glutamine, µg/ml sodium pyruvate and µg/ml nonessential aminoacid. U87 glioblastoma cell line was ordered from ATCC and was maintained in MEM medium with 10% fetal bovine serum with 1 µg/ml streptomycin.
Small Molecule Inhibitors
The small molecule inhibitor Y15 (called also inhibitor 14), 1,2,4,5-benzenetetraamine tetrahydrocloride has been described [3] and was ordered from Sigma Inc. Temozolomide was obtained from Sigma. Y15 was dissolved in DMSO at a concentration of 25 mM and stored at −20°C.
Antibodies
Polyclonal kinesin 14 antibody was obtained from Abcam, monoclonal tubulin and beta-Actin antibodies were obtained from Sigma.
RNA Isolation and Microarray Analysis
Expression profiling is accomplished using the HumanRef-8 whole-genome gene expression array and direct hybridization assay (Illumina, Inc.). Initially, 500ng total RNA was converted to cDNA, followed by in vitro transcription to generate biotin labeled cRNA using the Ambion Illumina Total Prep RNA Amplification Kit (Ambion, Inc.) according to the manufacturer’s instructions. The labeled probes were hybridized overnight at 58°C to the Illumina HumanRef-8 v3 Bead Chips. Following washing and staining with Cy3-streptavidin conjugate, the BeadChips were imaged using the Illumina Bead Array Reader to measure fluorescence intensity at each probe. Bead Chip data files were analyzed with Illumina’s Genome Studio gene expression module and Bioconductor package to determine gene expression signal levels. Briefly the raw intensity of Illumina Human ref-8 v3.0 gene expression array was scanned and extracted using Bead Scan, with the data corrected by background subtraction in Genome Studio module. The microarray data were submitted to NCBI with GEO accession number GSE43452.
Real-Time PCR
Real-time PCR with forward and reverse primers and fluorescent probe 5’FAM and 3’TAMPA was performed using isolated RNA, as described in [8]. Primer and probe sequences are available upon request. GAPDG was used as endogenous control. RQ was calculated for each gene tested from triplicate samples.
Bioinformatics and Statistical Analyses
The lumi module in the R-based Bioconductor package was used to transform the expression intensity to log2 scale. The log2 transformed intensity data were normalized using the Quantile normalization algorithm. The Limma program in the Bioconductor package under R computing environment was used to calculate the level of differential gene expression. For each comparison, we obtained the list of differentially expressed genes constrained by P-value <0.05 and at least 1.2 Fold change.
Western Blotting and Immunostaining
Western blotting and immunostaining was performed with kinesin antibody, as described [6].
RESULTS
Y15 Affects Expression of Common Genes that are Critical for Survival, Cell Cycle, Motility and Cytoskeleton Organization in DBTRG and U87 Glioblastoma Cells
To study the mechanism of Y15 in glioblastoma cells, we treated DBTRG and U87 cells with 10 µM Y15 for 24 hours and performed Illumina Human chip microarray analysis for the analyzing gene expression. In addition, we treated U87 cells with temozolomide 20 µM and combination of Y15 and temozolomide at the same doses for 24 hours. All samples were analyzed in duplicates. The structures and chemical name of Y15 (called also FAK inhibitor 14) and temozolomide are shown on Fig. (1A), upper panels. The heatmap of genes affected by Y15 in DBTRG and Y15, temozolomide and Y15 plus temozolomide in U87 cells are shown on Fig. (1A), lower left and right panels, respectively. Among 39694 gene probes that were analyzed 8034 genes were significantly changed (3834 up- and 4253 down-regulated) in DBTRG cells and 6555 genes changes (2737 up- and 3808 down-regulated) with p<0.05 in U87 cells, treated with Y15. The several up-regulated genes were validated by RT-PCR with gene-specific primers (Fig. 1B). The genes which were up-regulated by microarray analysis were up-regulated by RT-PCR in DBTRG cells (Fig. 1B, upper panel) and the same result was obtained in U87 cells (Fig. 1B, lower panel). The list of some important genes affected by Y15 in DGTRG cells are shown in Table 1. The significantly up-regulated genes (p<0.05) included Mdm-2, GADD45AA, PLK2 that play role in cell cycle arrest; TP53INP1, FAS,TNFAIP3, TXNIP which play role in apoptosis and phosphatases or dual specificity phosphatases PPP1R15A and DUSP5. The down-regulated genes were kinesins: KIF11, 14, 20A that play important role in motility (Table 1) and HSP90AA1, heat shock protein 90 that play role in heat-shock response. The significantly up and down-regulated genes, affected by Y15 in U87 cells are shown in Table 2. These genes also included genes that play role in phosphorylation, heat-shock response, apoptosis, cell cycle and cell motility.
Fig. (1). Y15 significantly affected gene expression in DBTRG and U87 cells by Illumina microarray analysis.
A. Upper panel: Y15 and Temozolomide structures. Lower panel: The heat map of up-(red) and down-regulated (green) genes in DBTRG cells, treated with Y15 (10 µM) for 24 hours and U87 cells treated with Y15 (10 µM), Temozolomide 20 µM and Y15+Temozolomide (TMZ) at the same doses for 24 hours are shown, p<0.05. B. The genes affected by micro-array were confirmed by RT-PCR analysis in DBTRG (upper panel) and U87 cells (lower panel). C. The genes affected by Y15 more than 1.5 fold in U87 and DBTRG cells and common genes are shown. There 237 genes were common among 1.5-fold affected genes in both cells.
Table 1.
List of several significantly up-regulated and down-regulated genes in Y15-treated DBTRG cells (p<0.05).
| Up-regulated genes | ||||
|---|---|---|---|---|
| Entrez | Symbol | Title | Fold Change | Function |
| 23645 | PPP1R15A | Protein phosphatase 1 | 17.52 | Dephosphorylate the regulatory subunit 15A translation initiation factor |
| 85707 | BEX2 | Brain-expressed X-linked 2 | 8.77 | Tumor suppressor |
| 1647 | GADD45AA | Growth arrest and DNA- damage-inducible GADD45A | 7.42 | Cell cycle; growth arrest; apoptosis |
| 54541 | DDIT4 | DNA-damage inducible transcript | 5.49 | Cell death |
| 7128 | TNFAIP3 | Tumor-necrosis factor, alpha induced protein 3 | 4.03 | Apoptosis |
| 2210 | HSPA6 | Heat shock 70 kDa protein 6 | 3.92 | Heat shock response |
| 144455 | E2F7 | E2F transcription factor 7 | 3.86 | Transcription factor |
| 1843 | DUSP1 | Dual specificity phosphatase 1 | 3.43 | Dual specificity phosphatase for ERK2 |
| 94241 | TP53INP1 | p53 inducible nuclear protein 1 | 3.03 | Apoptosis |
| 1847 | DUSP5 | Dual specificity phosphatase 5 | 2.48 | Dual specificity phosphatase 5 |
| 1028 | CDKN1A | Cyclin-dependent inhibitor 1A (p21,Cip1) | 2.46 | Growth arrest; cell cycle |
| 10769 | PLK2 | Polo-like kinase 2 | 2.11 | Cell cycle |
| 10628 | TXNIP | Thioredoxin interacting protein | 1.95 | Apoptosis; oxidation |
| 355 | FAS | FAS (TNF receptor superfamily) | 1.83 | Apoptosis |
| 4193 | Mdm2 | Mdm-2 p53 binding protein | 1.72 | Cell cycle, apoptosis |
| Down-regulated genes | ||||
| 54443 | ANLN | Anillin | 0.27 | Cell division |
| 890 | CCNA2 | Cyclin A2 | 0.33 | Cell cycle |
| 9928 | KIF14 | Kinesin family member 14 | 0.38 | Motility |
| 3832 | KIF11 | Kinesin family member 11 | 0.39 | Motility |
| 7153 | TOP2A | Topoisomerase II alpha | 0.39 | DNA topologic state |
| 10112 | KIF20A | Kinesin family member 20 | 0.44 | Motility |
| 3833 | KIFC1 | Kinesin family member c1 | 0.46 | Motility |
| 1111 | CHEK1 | CHK1 checkpoint homolog | 0.48 | Cell cycle |
| 983 | CDC2 | Cell division cycle 2 | 0.49 | Cell cycle |
| 3161 | HMMR | Hyaluronan-mediated motility receptor RHAMM (HMMR) | 0.51 | Motility; invasion |
| 4088 | SMAD3 | SMAD family receptor 3 | 0.57 | Transcription |
| 3320 | HSP90AA1 | Heat shock protein 90 kDa alpha | 0.58 | Heat shock response |
| 9578 | CDC42BPB | Cdc42 binding protein kinase beta | 0.61 | Cytoskeleton |
Table 2.
List of significantly up-regulated and down-regulated genes >1.5 fold in Y15-treated U87cells (p<0.05).
| Up-regulated genes | ||||
|---|---|---|---|---|
| Entrez | Symbol | Title | Fold Change | Function |
| 3310 | HSAP6 | Heat shock 70 kDa protein 6 | 5.52 | Heat-shock response |
| 3586 | IL10 | Interleukin 10 | 3.66 | Inhibits synthesisof cytokines |
| 3569 | IL6 | Interleukin 6 | 3.36 | Cytokine; B-cell differentiation |
| 1647 | GADD45A | Growth arrest and DNA- Damage inducible, alpha | 3.01 | Cell cycle; DNA damage |
| 84707 | BEX 2 | Brain expressed X-linked 2 | 2.65 | Apoptosis |
| 142679 | DUSP19 | Dual-specificity phosphatase 19 | 2.30 | Protein phosphatase |
| 1843 | DUSP1 | Dual-specificity phosphatase 1 | 2.00 | Protein phosphatase |
| 1847 | DUSP5 | Dual-specificity phosphatase 5 | 1.92 | Protein phosphatase |
| 1326 | MAP3K8 | Mitogen-activated protein kinase 8 | 1.89 | MAPK pathway |
| 581 | BAX | bcl-2-associated protein | 1.61 | Apoptosis |
| Down-regulated genes | ||||
| 5578 | PRKCA | Protein kinase C, alpha | 0.42 | Kinase |
| 7153 | TOP2A | Topoisomerase II alpha | 0.45 | DNA topological Structure |
| 214 | ALCAM | Leucocyte cell adhesion molecule (ALCAM) | 0.56 | Adhesion, migration |
| 9585 | KIF20B | Kinesin family member 20B | 0.57 | Motor enzyme for Cytokinesis |
| 3673 | ITGA2 | Integrin, alpha 2 | 0.59 | Adhesion, attachent |
| 3832 | KIF11 | Kinesin family member 11 | 0.60 | Movement of chromosomes |
| 899 | CCNF | Cyclin F | 0.61 | Cell cycle |
| 3667 | IRS1 | Insulin receptor substrate 1 | 0.63 | Insulin-like growth Factor receptor substrate |
| 3309 | HSPA5 | Heat shock 70kDa protein 5 | 0.64 | Heat shock response |
| 142 | PARP1 | Poly(ADH-ribose) polymerase | 0.66 | Chromatin structure, family member 1 base excision repair |
Microarray Analysis Detected 232 Common Genes Affected by Y15 in two Glioblastoma Cell Lines
There were 1332 and 462 genes that were up or down-regulated in DBTRG and U87 cells, respectively more than 1.5 fold, p<0.05 (Fig. 1C) and there were 237 of all genes common genes between cell lines. The representative common genes affected by Y15 are shown in Table 3. The common genes up-regulated >1.5 fold by Y15 in both cell lines included BEX-2, brain expressed X-linked 2 gene; GADD45A, HSPA6 (heat-shock 70); DUSP1, DUSP 5 (dual-phosphatases); CDKN1A (p21) (Table 3) and common down-regulated genes kinesins, BUB1, PARP1, POLA1, etc (Table 3).
Table 3.
The list of several common and significantly up-regulated and down-regulated genes (>1.5 fold) in Y15-treated DBTRG and U87cells, p<0.05.
| Up-regulated genes | |||||
|---|---|---|---|---|---|
| Entrez | Symbol | Title | Fold Change | Function | |
| DBTRG | U87 | ||||
| 84707 | BEX2 | Brain expressed X-linked 2 | 8.77 | 2.65 | Apoptosis |
| 1647 | GADD45A | Growth arrest and DNA damage | 7.42 | 3.01 | DNA damage |
| 3310 | HSPA6 | Heat shock 70 kDa protein 6 | 3.92 | 5.52 | Heat shock |
| 1843 | DUSP1 | Dual specificity phospatase 1 | 3.42 | 2.00 | Phosphatase |
| 4084 | MXD1 | MAX dimerization protein | 2.81 | 1.52 | Transcription |
| 1847 | DUSP5 | Dual specificity phospatase 5 | 2.49 | 1.94 | Phosphatase |
| 1026 | CDKN1A | Cyclin-dependent kinase inhibitor1A, (p21, Cip 1) | 2.45 | 2.22 | Cell cycle |
| 64112 | MOAP1 | Modulator of apoptosis 1 (MOAP1) | 1.99 | 1.55 | Apoptosis |
| 56271 | BEX4 | BEX family member 4 | 1.67 | 1.69 | BEX family |
| 3606 | IL18 | Interleukin 18 | 1.54 | 2.76 | Cytokine |
| Down-regulated genes | |||||
| 9928 | KIF 14 | Kinesin family member 14 | 0.38 | 0.61 | Movement of chromosomes |
| 7153 | TOP2A | Topoisomerase II alpha | 0.39 | 0.44 | Controls topologic state of DNA |
| 7153 | KIF 11 | Kinesin family member 11 | 0.39 | 0.60 | Movement of chromosomes |
| 10112 | KIF20A | Kinesin family member 20A | 0.44 | 0.65 | Movement of chromosomes |
| 699 | BUB1 | Budding inhibited by benzimidazoles 1 | 0.45 | 0.54 | Cell cycle |
| 899 | CCNF | Cyclin F (CCNF) | 0.50 | 0.60 | Cell cycle |
| 9585 | KIF20B | Kinesin family member 20B | 0.52 | 0.57 | Movement of chromosomes |
| 10635 | RAD51AP1 | RAD51 associated protein 1 | 0.55 | 0.63 | Double stand break repair |
| 142 | PARP1 | Poly(ADP-ribose) polymerase Family | 0.56 | 0.66 | Base excision repair |
| 8914 | TIMELESS | Timeless Drosophila homolog | 0.57 | 0.67 | RNA synthesis |
| 5422 | POLA 1 | Polymerase (DNA-directed) alpha 1 | 0.60 | 0.64 | DNA replication |
| 7171 | TPM4 | Tropomyosin 4 | 0.63 | 0.63 | Cell movement |
Y15 Decreased Expression of Kinesins in both Glioblastoma Cell Lines
We detected many kinesins which were down-regulated by Y15 in both glioblastoma cell lines (Table 4). There were many common down-regulated kinesins KIF11, KIF14, KIF20A, KIF20B and some were specific to DBTRG cell line: KIF2C, KIFC1, KIF23 (Table 4). We performed Western blotting in Y15-treated DBTRG cells and detected decreased expression of KIF14 kinesin in a dose-dependent manner (Fig. 2A). In addition, we performed immunostaining of Y15-treated DBTRG cells and detected decreased expression of Kinesin 14 and changes of its localization from cytoplasmic homogenous to nuclear dotted localization which was different from control tubulin (Fig. 2B). Since recent report demonstrated that down-regulation of kinesin 14 increased binucleated cell formation [9], we performed Hoechst staining of Y15-treated DBTRG cells and found that Y15 caused decrease of kinesin 14 and also caused bi-nucleated cells (Fig. 2C). This shows that down-regulation of FAK with Y15 decreases kinesin 14 expression, localization and affects its cellular functions. In summary, Y15 decreased expression of many kinesins in two glioblastoma cells, changed its localization and affected its cell function suggesting that FAK and kinesin functions are linked.
Table 4.
List of significantly up-regulated and down-regulated kinesin genes in Y15-treated DBTRG and U87 cells (p<0.05).
| Entrez | Symbol | Title | Fold Change | Function |
|---|---|---|---|---|
| Down-regulated genes by Y15 in DBTRG cells | ||||
| 11004 | KIF2C | Kinesin family member 2C | 0.66 | Movement organelles, Microtubules, mitosis |
| 56992 | KIF15* | Kinesin family member 15 | 0.54 | Movement organelles, Microtubules, mitosis |
| 9493 | KIF23 | Kinesin family member 23 | 0.52 | Movement organelles, Microtubules, mitosis |
| 9585 | KIF20B | Kinesin family member 20B | 0.52 | Movement organelles, |
| 3833 | KIFC1 | Kinesin family member C1 | 0.46 | Movement organelles, Microtubules, mitosis |
| 10112 | KIF20A | Kinesin family member 20A | 0.43 | Movement organelles microtubules, mitosis |
| 24137 | KIF4A | Kinesin family member 4 A | 0.41 | Movement organelles microtubules, mitosis |
| 3832 | KIF11 | Kinesin family member 11 | 0.39 | Movement organelles microtubules, mitosis |
| 9928 | KIF14 | Kinesin family member 14 | 0.37 | Movement organelles microtubules, mitosis |
| Down-regulated genes by Y15 in U87 cells | ||||
| 9585 | KIF20B | Kinesin family member 20B | 0.57 | Movement organelles |
| 10112 | KIF20A | Kinesin family member 20A | 0.65 | Movement organelles microtubules, mitosis |
| 9928 | KIF14 | Kinesin family member 14 | 0.61 | Movement organelles microtubules, mitosis |
| 3832 | KIF11 | Kinesin family member 11 | 0.60 | Movement organelles microtubules, mitosis |
| Down-regulated genes by Y15+TMZ in U87 cells | ||||
| 56992 | KIF15* | Kinesin family member 15 | 0.65 | Movement organelles microtubules, mitosis |
| 10112 | KIF20A | Kinesin family member 20A | 0.43 | Movement organelles microtubules, mitosis |
| 3832 | KIF11 | Kinesin family member 11 | 0.59 | Movement organelles microtubules, mitosis |
| 9928 | KIF14 | Kinesin family member 14 | 0.61 | Movement organelles microtubules, mitosis |
| 9585 | KIF20B | Kinesin family member 20B | 0.57 | Movement organelles microtubules, mitosis |
Underlined are common genes down-regulated by Y15 in DBTRG and U87 cells by Y15 and Y15+TMZ;
common genes down-regulated in DBTRG cells by Y15 and in U87 cells by Y15+TMZ
Fig. (2). Y15 decreases expression of kinesin 14 in DBTRG glioblastoma cells.
A. DBTRG cells were treated with different doses of Y15 for 24 hours, and Western blotting was performed with kinesin 14 antibody. Western blotting with beta-actin showed equal protein loading. The structure of Y15 is shown. B. Immunostaining shows decrease of kinesin expression and change of its localization in Y15-treated DBTRG cells. Cells were immunostained with primary kinesin 14 antibody, then with FITC-conjugated secondary anti-rabit antibody (green) and tubulin primary and secondary Texas-Red anti-mouse antibody (red). Nuclei were stained with Hoechst. Merged image is shown on the right panel. C. Hoechst staining of Y15-treated cells demonstrates increase of bi-nucleated cells. Arrows show binucleated cells.
Microarray Analysis Detects Differentially Expressed Genes Affected by Temozolomide and by Combination of Y15 and Temozolomide in Glioblastoma Cells
Since recently we detected that combination of Y15 and temozolomide significantly decreased viability and tumor growth of glioblastoma cells [6], we performed analysis of genes affected by temozolomide, and combination of temozolomide (TMZ) and Y15 versus untreated cells in U87 cell line. The genes that were >1.5 fold up and down-regulated in Temozolomide (TMZ)-treated U87 cells are shown in Table 5. The up-regulated genes by TMZ included CDKN1A, cyclin-dependent kinase inhibitor 1A (p21, Cip); TXNIP, thioredoxin-interacting protein; EGFBP3, insulin-like growth factor binding protein; and down-regulated was SerpinB2, serpin peptidase inhibitor B member 2. We also found set of genes that were significantly up or down-regulated by combination of Y15 plus TMZ-treated cells versus untreated cells (p<0.05) (Table 6). Some of these genes are shown in Table 5 and include up-regulated HSPA6, BEX2, DUSP-5 and down-regulated COL1A1 (collagen, type I) or GNAI2) guanine nucleotide-binding protein. Several of the genes affected by combination of Y15 and TMZ were more affected than by each inhibitor alone. Among genes significantly up-regulated in Y15 and TMZ-treated cells, but not in Y15 or TMZ-treated cells were: cytochrome c oxidase subunit VIIb, COX7B; interferon, gamma-inducible protein 16, IFI16; DNA-damage-inducible transcript 4, DDIT; growth arrest and DNA damage-inducible, GADD45G and others. Among down-regulated genes were: ABL, AKT1, JAK1, ALDH1A3, Gli3, which are known survival factors (Table 6, marked by asterisk). Thus, analysis of gene expression in Y15 or Y15 plus Y15-treated cells identifies genes affected by Y15 in both glioblastoma cells, by temozolomide and by combination of Y15 and temozolomide, which is important for understanding mechanism of downstream signaling and resistance in response to FAK inhibitor Y15.
Table 5.
List of significantly up-regulated and down-regulated genes >1.5 fold in Temozolomide-treated U87cells (p<0.05).
| Up-regulated Genes | ||||
|---|---|---|---|---|
| Entrez | Symbol | Title | Fold Change | Function |
| 1026 | CDKN1A | Cyclin-dependent kinase inhibitor 1A (p21, Cip1) | 1.82 | Cell cycle inhibitor |
| 10628 | TXNIP | Thioredoxin interacting protein TXNIP | 1.62 | Thioredoxin inhibitor; tumor suppressor |
| 3486 | IGFBP3 | Insulin-like growth factor binding protein 3 (IGFBP3) | 1.61 | Cell growth |
| Down-regulated genes | ||||
| 5055 | SerpinB2 | Serpin peptidase inhibitor, clade B, | 0.57 | Inhibition of member 2 apoptosis |
Table 6.
List of several significantly up-regulated genes in U87 cells treated with Y15+temozolomide (TMZ) versus untreated (p<0.05).
| Up-regulated genes | ||||
|---|---|---|---|---|
| Entrez | Symbol | Title | Fold Change vs Untreated | Function |
| 3310 | HSPA6 | Heat shock 70 kDa protein 6 | 6.64 | Heat shock |
| 1649 | DDIT3 | DNA-damage-inducible transcript 3 | 4.74 | Inhibits activity of C/EBP factor |
| 3576 | IL-8 | Interleukin- -8 | 3.57 | Cytokine |
| 84707 | BEX2 | Brain-expressed X-linked 2 | 3.13 | Apoptosis |
| 1847 | DUSP 5 | Dual-specificity kinase 5 | 2.30 | Phosphatase |
| 8660 | IRS2 | Insulin receptor substrate 2 | 2.01 | Insulin growth factor receptor |
| 1349 | COX7B* | Cytochrome c oxidase subunit VIIb | 1.52 | Electron transport |
| 3428 | IFI16* | Interferon, gamma-inducible protein 16 | 1.31 | Transcriptional repressor |
| 54541 | DDIT4* | DNA-damage-inducible transcript 4 | 1.30 | Inhibits cell growth |
| 1586 | CYP17A1* | Cytochrome P450, family 17, subfamily A | 1.27 | Electron transport |
| 10912 | GADD45G* | Growth arrest and DNA damage-inducible | 1.24 | DNA damage |
| 11266 | DUSP12* | Dual-specificity phosphatase 12 | 1.20 | Phosphatase |
| 2501 | FTHL8* | Ferritin, heavy polypeptide-like 8 | 1.17 | Iron storage |
| Down-regulated genes | ||||
| 11127 | KIF3A* | Kinesin family member 3A | 0.88 | Movement of chtomosomes |
| 25 | ABL* | c-abl oncogene | 0.87 | Oncogene |
| 207 | AKT1* | v-akt murine thymoma viral oncogene homolog 1 | 0.87 | Survival |
| 3835 | KIF22 | Kinesin 22 | 0.86 | Movement of chromosomes |
| 1739 | DLG1* | H.s Disks large homolog1 (Drosophila) | 0.84 | Development |
| 31 | ACACA* | Acetyl-coenzyme A Carboxylase alpha | 0.81 | Metabolism |
| 3716 | JAK1* | Janus kinase 1, JAK1 | 0.81 | Kinase, interferon pathway |
| 2737 | GLI3* | GLI-Kruppel family member Gli3 | 0.80 | Transcriptional activator/repressor of Sonic Hedgehoc pathway |
| 220 | ALDH1A3* | Aldehyde dehydrogenase 1 family member A3 | 0.80 | Detoxification of aldehyde |
| 2771 | GNAI2 | Guanine nucleotide binding protein | 0.63 | Guanine nucleotide- binding protein |
| 1277 | COL1A1 | Collagen, type I, alpha 1 | 0.58 | Extracellular Matrix signaling |
significant up- or down-regulation (p<0.05) in Y15+TMZ-treated cells, but not in Y15 or TMZ-treated cells
DISCUSSION
This report demonstrates gene profiling in response to FAK inhibitor Y15 in glioblastoma cells. The Illumina microarray analysis demonstrated 8087 and 6555 genes affected by Y15 among 34,694 genes analyzed. There were 1332 and 462 genes that were >1.5 fold affected by Y15 and 237 common genes were discovered in DBTRG and U87 cells. The combination of Y15 and temozolomide detected specific genes that were more significantly affected by this combination compared with each drug alone in U87 cells.
In Y15-treated DBTRG glioblastoma cells, some genes, which were up-regulated included: Mdm-2, GADD45AA, PLK2, TXNIP, DUSP1 and DUSP5 in DBTRG cells (p<0.05). Mdm-2, GADD45AA, PLK2 are important genes that regulate and mediate cell cycle arrest. TXNIP or thioredoxin interacting protein play a crucial role in apoptosis and oxidation [10]. DUSP1 and DUSP5 are Dual Specificity Phosphatases, which can dephosphorylate both phosphotyrosine and phosphoserine/phosphotreonines in the same substrate and are involved in many signaling cellular pathways such as Mitogen Activated Protein Kinase (MAPK) pathways [11, 12]. Some of the genes which were down-regulated by Y15 in DBTRG cells included HSP90AA1 and different members of the Kinesin family: KIF11, KIF14, and KIF20A. The HSP90AA1 encodes a heat shock protein 90, which is important in mediating the protective heat-shock response in cancer cells. In U87 glioblastoma cells treated with Y15 there were many deregulated genes which play important roles in cell cycle arrest, phosphorylation, cell motility, and heat-shock response.
Kinesin family members are important for cell motility, mitotic function, spindle and microtubule formation. The massive down-regulation of kinesins by FAK inhibitors suggests that there is important role of FAK in motility of chromosomes. It is known that kinesins play significant role in microtubules, mitosis and chromosome movement and can be therapeutic targets in cancer [13]. Especially kinesin 14 has been found to play a key role in microtubule and kinetochore function and was often overexpressed in different types of cancer [14, 15]. Down-regulation of kinesin 14 with siRNA induced cytokinesis failure and caused bi-nucleated nuclei [9]. We found that FAK changed localization from cytoplasm to the nucleus and that kinesin changed their localization in Y15-treated cells that suggests a novel function of FAK in the nucleus, associated with microtubule movement and mitotic function. There were previous reports on FAK role in gene regulation, for example FAK up-regulated cyclin D4 and interacted with p53 in the nucleus [16, 17]. These data support the role of FAK in the nucleus and data on gene expression provide a basis for novel mechanism of Y15 in glioblastoma cells.
Microarray analysis detected that Y15-treated DBTRG and U87 cells shared 237 genes common, which were more than 1.5 fold up- or down-regulated by Y15 and represent 20% and 51% of all affected genes in these cells, respectively. The data demonstrate that Y15 affects common signaling pathways. Among the common up-regulated genes were DUSP1 and DUSP5, known as dual-phosphatases. There were also BEX-2 and BEX-4, brain expressed X-linked gene that can play a significant role in apoptosis. Among down-regulated genes there were several kinesins. There were several common kinesins affected in DBTRG and U87 cell lines and in Y15 plus temozolomide U87 and Y15-treated U87 cells, while some were cell line-specific. The massive down-regulation of kinesins in response to FAK inhibitor Y15 suggests that there is a cross-linked signaling between FAK and kinesin functions. Since there were reports on drug-resistance associated with several kinesins and kinesin-related proteins [18, 19], their significant down-regulation by Y15 and by combination of Y15 and temozolomide is important for efficient inhibition of glioblastoma tumor growth and development of future targeted therapeutics. We detected decreased expression of kinesins and increase in formation of binucleated cells in Y15-treated glioblastoma. This cross-linked signaling also confirms nuclear functions of FAK.
We detected genes that were up-regulated in response to Y15 and temozolomide more significantly than in response to each inhibitor alone: Cox7B; interferon, gamma-inducible transcript 4; cytochrome P450; growth arrest and DNA damage-inducible, GADD45G, which is consistent with decreased viability of U87 cells, treated with Y15 and temozolomide [6]. Among down-regulated genes in response to Y15 and temozolomide were KIF3A; AKT1; JAK1; GLI3 and ALDH1A3, known to play important role in survival and cancer stem cell biology.
Thus, these data demonstrate for the first time genes affected by FAK inhibitor Y15 and by combination of Y15 and temozolomide. The gene profile of these genes is important for developing of FAK-targeted therapy and combination therapy for glioblastoma.
ACKNOWLEDGEMENTS
We would like to acknowledge Susan Komen for the Cure Foundation grant (VMG) and NCI RO-1 grants (WGC) for support of these studies.
Footnotes
CONFLICT OF INTEREST
Dr. Vita Golubovskaya discloses that she is co-founders and stock holder of CureFAKtor Pharmaceuticals.
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