13 research outputs found
ALK signaling cascade confers multiple advantages to glioblastoma cells through neovascularization and cell proliferation
<div><p>Anaplastic lymphoma kinase (ALK), which is a receptor tyrosine kinase, is essentially and transiently expressed in the developing nervous system. Here we examined the functional role of the <i>ALK</i> gene in glioblastomas (GBMs). In clinical samples of GBMs, high ALK expression without gene rearrangements or mutations was frequently observed in perivascular lesions, in contrast to the relatively low expression in the perinecrotic areas, which was positively correlated with N-myc and phosphorylated (p) Stat3 scores and Ki-67 labeling indices. ALK immunoreactivity was also found to be associated with neovascular features including vascular co-option and vascular mimicry. In astrocytoma cell lines, cells stably overexpressing full-length ALK showed an increase in expression of pStat3 and pAkt proteins, as well as hypoxia-inducible factor-1Ī± (HIF-1Ī±) and vascular endothelial growth factor-A (VEGF-A) mRNAs, in contrast to cells with knockdown of endogenous ALK which showed decreased expression of these molecules. Transfection of the constitutively active form of Stat3 induced an increase in <i>HIF-1</i>Ī± promoter activity, and the overexpression of HIF-1Ī± in turn resulted in enhancement of <i>VEGF-A</i> promoter activity. In addition, cells with overexpression or knockdown of ALK also showed a tendency toward increased and decreased proliferation, respectively, through changes in expression of pAkt and pStat3. Finally, <i>ALK</i> promoter was significantly activated by transfection of Sox4 and N-myc, which are known to contribute to neuronal properties. These findings therefore suggest that N-myc/Sox4-mediated ALK signaling cascades containing Stat3, Akt, HIF-1Ī±, and VEGF-A confer multiple advantages to tumor growth through alterations in neovascularization and cell proliferation in GBMs.</p></div
ALK mRNA expression and gene arrangement status in GBMs.
<p>(A) Staining by hematoxylin and eosin (HE) and ISH for ALK mRNA. Note the abundant mRNA signals in tumor cells around vascular components (indicated by arrows), in contrast to the weak signals adjacent to necrotic foci (partitioned by dotted line). Insets show magnified views of the boxed areas. Original magnification, x100 and x400 (inset). (B) Relationship of ALK (5A4) expression between the ISH signal positivity and the IHC score in GBMs. (C) FISH analysis of four GMB cases with high ALK expression. The interphase nuclei of these cases indicate absence of ALK rearrangement, as shown by the merged red and green signals (indicated by arrows).</p
Correlations among IHC markers investigated in GBM cases.
<p>Correlations among IHC markers investigated in GBM cases.</p
Additional file 1: Figure S1. of Cooperation of Sox4 with Ī²-catenin/p300 complex in transcriptional regulation of the Slug gene during divergent sarcomatous differentiation in uterine carcinosarcoma
(A) Ishikawa (Ish) cells were transfected with Sox4, Sox7, and Sox9 reporter constructs, together with either Sox4, Sox7, or Sox9. Relative activity was determined based on arbitrary light units of luciferase activity normalized to pRL-TK activity. The activities of the reporter plus the effector relative to that of the reporter plus empty vector are shown as meansāĀ±āSDs. The experiment was performed in duplicate. (B) Western blot analysis for the indicated proteins from Ishikawa, Hec251, and Hec6 cells cultured in STK2. (C) Western blot analysis for the indicated proteins from H6SL#8 and mock cells. (TIF 2816 kb
Relationship of ALK expression with Sox4 and N-myc in astrocytomas.
<p>(A) Various <i>ALK</i> promoter constructs used in this study. (B) KS-1 cells were transfected with <i>ALK</i> promoter constructs, together with the indicated <i>Sox</i> genes. Relative activity was determined based on arbitrary light units of luciferase activity normalized to pRL-TK activity. The activities of the reporter plus the effector relative to that of the reporter plus empty vector are shown as meansĀ±SDs. The experiment was performed in duplicate. (C) KS-1 cells were transfected with <i>ALK</i> promoter constructs, together with either Sox4, c-myc, or N-myc. (D) Various promoter constructs were used for evaluating transcriptional regulation of the <i>ALK</i> promoter by either Sox4 or N-myc. (E) The shortest <i>ALK</i> promoter constructs containing mutations in two putative E-boxes (E1 and E2), along with either Sox4 or N-myc, were transfected into KS-1 cells. (F) The Sox4 (left) and the N-myc (right) promoter constructs, along with either c-myc, N-myc, or Sox4, were transfected into KS-1 cells. (G) Left: staining by hematoxylin and eosin (HE) and IHC for N-myc and c-myc in GBMs. Note the strong immunoreactivity for N-myc, but not c-myc, in tumor cells around vessel (indicated by arrows) but not perinecrotic area (necrotic lesion is partitioned by dotted line). Right: IHC scores for N-myc and c-myc in perivascular (Peri-v) and perinecrotic (Peri-n) lesions.</p
Association between ALK expression and cell proliferation in astrocytoma cells.
<p>(A) Left: KS-ALK#4 cells and mock cells were seeded at low density. The cell numbers are presented as meansĀ±SDs. P0, P2, P4, P6, and P8 indicate 0, 2, 4, 6, and 8 days after cell passage, respectively. Right: Cell Counting Kit-8 (CCK-8) assay for cell proliferation. Cells were seeded at 1x10<sup>3</sup> cells in 96-well plates. Viable cell numbers were quantitated. Relative absorbance values (P5 or P7 relative to P2) are presented as meansĀ±SDs. P2, P5, and P7 indicate 2, 5, and 7 days after cell passage, respectively. This experiment was performed in triplicate using independent samples. (B) Western blot analysis for the indicated proteins in ALK#4 and mock cells after serum stimulation for the times shown. (C) Left: KINGS-shALK#37, #46 cells, and mock cells were seeded at low density. The cell numbers are presented as meansĀ±SDs. P0, P3, P6, and P9 indicate 0, 3, 6, and 9 days after cell passage, respectively. Right: CCK-8 assay for cell proliferation to quantitate viable cell numbers as mentioned above. (E) Staining by hematoxylin and eosin (HE) and IHC for pStat3, pAkt, and Ki-67 in GBMs. Note the strong immunoreactivity for these molecules in perivascular lesions (vessels are indicated by arrows), in contrast to the weak immunoreaction in perinecrotic areas (necrotic lesion is partitioned by dotted line). Original magnification, x100. (F) IHC scores for pStat3 and pAkt and Ki-67 labeling indices in perivascular (Pv) and perinecrotic (Pn) lesions.</p
Up-regulation of ALK expression in hypervascular areas of GBMs.
<p>(A) Left: staining by hematoxylin and eosin (HE) and IHC for ALK in GBMs. Note the strong cytoplasmic ALK (5A4) positivity in cells around vascular components (a) (indicated by arrows) of GBMs, in contrast to the sporadic staining or absence in tumor cells adjacent to necrotic foci (b) (partitioned by dotted line). Panels (a) and (b) are magnified views of the boxed areas. Original magnification, x100 (left) and x200 (middle and right). Right: IHC score for ALK (5A4) in perivascular (Peri-v) and perinecrotic (Peri-n) areas. (B) Staining by HE (upper and lower left) and double-staining for ALK/CD34 (upper and lower middle) and SMA/CD34 (upper and lower right) in mature vessels (upper) and microvessels (lower) in GBM. Strong ALK (5A4) (brown) positivity is observed in GBM cells surrounding CD34 (red)-positive mature vessels (upper middle), while focal ALK (5A4) immunoreactivity is also evident in CD34-positive microvessels (lower middle). Note the close association between SMA- and CD34-positive cells in both tumor vasculatures (upper and lower left). Insets show magnified views of the boxed areas. Original magnification, x200 and x400 (inset). (C) Left: staining by HE and IHC for ALK and CD34 in GBMs. Note the strong ALK (5A4) immunopositivity in areas with high CD34 immunoreactivity, in contrast to the sporadic staining or absence in the low immunoreactivity lesions. Original magnification, x100. Right: relationship between ALK (5A4) immunointensity and microvascular density as determined by CD34 immunoreactivity in GBMs. The data shown are meansĀ±SDs. (D) Western blot analysis of the indicated proteins after CoCl<sub>2</sub> treatment with the different doses shown for 24 hours (left) and 50 Ī¼M CoCl<sub>2</sub> for the time shown (right) in KINGS-1 cells.</p
Up-regulation of ALK expression in GBMs.
<p>(A) Staining by hematoxylin and eosin (HE) and IHC for ALK in astrocytomas. Note the diffuse distribution of strong cytoplasmic ALK (5A4)-positive cells in grade IV (GBM) tumor, in contrast to the sporadic distribution or absence in grade II and III astrocytomas. Insets show the magnified views of the boxed area. Original magnification, x100 and x400 (inset). (B) IHC score for ALK (5A4) in astrocytomas. G, grade (C) Relationship of ALK (5A4) expression with overall survival and progression-free survival for all grades of astrocytomas. n, number of cases. (D) Relationship of ALK (5A4) expression and <i>isocitrate dehydrogenase 1 (IDH1)</i> gene status with overall survival and progression-free survival for all grades of astrocytomas. n, number of cases. wt, wild type; mt, mutant type.</p
Relationship between ALK expression and neovascularization in GBMs.
<p>Relationship between ALK expression and neovascularization in GBMs.</p
Additional file 3: Figure S3. of Cooperation of Sox4 with Ī²-catenin/p300 complex in transcriptional regulation of the Slug gene during divergent sarcomatous differentiation in uterine carcinosarcoma
(A) Hec251 (left) and Hec6 cells (right) were transfected with Slug reporter constructs, together with Ī²-cateninĪS45 (Ī²-cat), Sox4, and p300. Relative activity was determined based on arbitrary light units of luciferase activity normalized to pRL-TK activity. The activities of the reporter plus the effector relative to that of the reporter plus empty vector are shown as meansāĀ±āSDs. The experiment was performed in duplicate. (B) After transfection of HA-Ī²-cateninĪS45 and GFP-Sox4, cells were stained with anti-HA antibody. Immunopositive cell is indicated by arrow. Nuclei were stained by DAPI. (C,D) Hec251 (C) and Hec6 (D) cells were transfected with pM-Ī²-catenin (left) or pM-Sox4 (right), along with pGL5 luc, Sox4, Sox7, Sox9, and p300. The experiment was performed in duplicate. (TIF 2017 kb