PTK7 regulates Id1 expression in CD44-high glioma cells

Abstract

Background: CD44 is a molecular marker associated with molecular subtype and treatment resistance in glioma. More effective therapies will result from approaches aimed at targeting the CD44-high gliomas.

Methods: Protein tyrosine kinase 7 (PTK7) mRNA expression was analyzed based on The Cancer Genome Atlas glioblastoma dataset. PTK7 expression was depleted through lentivirus-mediated short hairpin RNA knockdown. Terminal deoxynucleotidyl transferase dUTP nick-end labeling was used to evaluate cell apoptosis following PTK7 knockdown. Gene expression analysis was performed on Affymetrix microarray. A nude mice orthotopic tumor model was used to evaluate the in vivo effect of PTK7 depletion.

Results: PTK7 is highly expressed in CD44-high glioblastoma and predicts unfavorable prognosis. PTK7 knockdown attenuated cell proliferation, impaired tumorigenic potential, and induced apoptosis in CD44-high glioma cell lines. Gene expression analysis identified inhibitor of DNA Binding 1 (Id1) gene as a potential downstream effector for PTK7. Overexpression of Id1 mostly restored the cell proliferation and colony formation attenuated by PTK7 depletion. PTK7 enhanced anchorage-independent growth in normal human astrocytes, which was attenuated by Id1 knockdown. Furthermore, PTK7 regulated Id1 expression through modulating TGF-β/Smad signaling, while pharmacological inhibition on TGF-β/Smad signaling or PTK7/Id1 depletion attenuated TGF-β-stimulated cell proliferation. PTK7 depletion consistently reduced Id1 expression, suppressed tumor growth, and induced apoptosis in a murine orthotopic tumor model, which could be translated into prolonged survival in tumor-bearing mice.

Conclusions: PTK7 regulates Id1 expression in CD44-high glioma cell lines. Targeting PTK7 could be an effective strategy for treating glioma with high CD44 expression.

Keywords: CD44; PTK7; cell proliferation; glioma; tumorigenesis.

Fig. 1.

PTK7 is highly expressed in CD44-high GBMs and predicts poor prognosis. (A) PTK7 mRNA expression in nontumor brain tissues and gliomas (astrocytoma grade II, III; GBM; oligodendrocytoma grade II, III) based on GSE4290 dataset (http://www.ncbi.nlm.nih.gov/sites/GDSbrowser?acc=GDS1962). (B) Survival analysis on PTK7 expression in TCGA GBM dataset. (C) PTK7 is highly expressed in TCGA mesenchymal GBM subclass. (D) PTK7 and CD44 protein expression in mouse brain and human GBM tissue lysates. (E) Immunostaining on PTK7 and CD44 in GBM03 frozen tissue section. Bars: 100 μm.

Fig. 2.

(A) Expression of PTK7 and CD44 in human GBM cell lines. (B) PTK7 knockdown attenuated glioma cell proliferation in LN18, SF295, and T98G cells. *P < .05, as compared with scramble (Scr) control. (C) PTK7 knockdown reduced BrdU incorporation in LN18, SF295, and T98G cells. *P < .05, as compared with scramble (Scr) control. (D) PTK7 knockdown impaired soft agar colony formation in LN18, SF295, and T98G cells. *P < .05, as compared with scramble (Scr) control. (E and F) PTK7 knockdown induced apoptosis in LN18, SF295, and T98G cells. Bars: 50 μm. *P < .05, as compared with scramble (Scr) control. (G) PTK7 knockdown decreased tumorsphere formation in glioma initiating cell lines GIC-7 and GIC-7. *P < .05, as compared with scramble (Scr) control. (H) PTK7 knockdown reduced BrdU incorporation in glioma initiating cell lines GIC-7 and GIC-7. *P < .05, as compared with scramble (Scr) control.

Fig. 3.

Id1 is a potential downstream effector of PTK7 signaling. (A) PTK7 knockdown altered gene expression in LN18, SF295 and GIC-7 glioma cells. (B) PTK7 depletion attenuated Id1 mRNA and protein expression in LN18, SF295 and GIC-7 glioma cells. *P < .05, as compared with scramble (Scr) control. (C) Id1 knockdown attenuated glioma cell proliferation in LN18, SF295, and GIC-7 glioma cells. *P < .05, as compared with scramble (Scr) control. (D) Id1 knockdown impaired soft agar colony formation in LN18, SF295, and GIC-7 glioma cells. *P < .05, as compared with scramble (Scr) control. (E) Id1 knockdown induced apoptosis in LN18, SF295, and GIC-7 glioma cells. *P < .05, as compared with scramble (Scr) control. (F–H) Overexpression of Id1 rescued cell proliferation and colony formation attenuated by PTK7 depletion. *P < .05, as compared with scramble (Scr) control. **P < .05, as compared with shPTK7 group.

Fig. 4.

PTK7 up-regulates Id1 to induce anchorage-independent growth in normal human astrocytes. (A) Overexpression of PTK7 upregulated Id1 expression in normal human astrocytes. (B) PTK7 promoted anchorage-independent growth in normal human astrocytes. *P < .05, as compared with GFP control. Bars: 100 μm. (C and D) Overexpression of Id1 promoted anchorage-independent growth in normal human astrocytes. *P < .05, as compared with GFP control. (E and F) Knockdown of Id1 attenuated PTK7-induced anchorage-independent growth in normal human astrocytes. *P < .05, as compared with GFP control. **P < .05, as compared with PTK7-expressing group.

Fig. 5.

PTK7 regulates Id1 expression via TGF-β/Smad pathway. (A) TGF-β receptor inhibition attenuated PTK7-induced Id1 expression in A172 glioma cells. After PTK7 transfection, A172 cells were incubated with various inhibitors for PDGFRα (imatinib, 5 μM), Tarceva (EGFR, 5 μM), TGF-β receptor inhibitor (SB413542, 10 μM), MEK/ERK (PD98059, 10 μM) for 24 hours. (B) PTK7 regulates Id1 expression via TGF-β/Smad pathway. After serum starvation for 36 hours, glioma cells (Scr or shPTK7) were treated with TGF-β (10 ng/mL) for 24 hours. SB413542 was used as a positive control for the blockade of TGF-β signaling. (C) PTK7/Id1 knockdown attenuated TGF-β-induced cell proliferation in glioma cell lines LN18 and SF295. *P < .05, as compared with no-TGF-β control. **P < .05, as compared with TGF-β-stimulated group. (D) SB431542 effects on PTK7-expressing or PTK7-depleted glioma cells. The number of viable cells in PTK7-expressing or PTK7-depleted cells without SB431542 treatment was regarded as 100%. *P < .05, as compared with no drug control in PTK7-expressing glioma cells.

Fig. 6.

Targeting PTK7 attenuates in vivo tumor growth and prolongs tumor-bearing mice survival. (A) PTK7 depletion attenuated subcutaneous tumor growth. *P < .05, as compared with scramble (Scr) control. (B) Western blotting analysis on tumor lysates from subcutaneous tumor with PTK7 depletion at day 30 after implantation. (NB, normal mouse brain lysate). (C) Targeting PTK7 prolongs tumor-bearing mice survival in an orthotopic xenograft model. (D) PTK7 depletion attenuates in vivo tumor growth in an orthotopic xenograft model. *P < .05, as compared with scramble (Scr) control. Bars: 1 mm. (E) Immunostaining of the intracranial tumors at day 25 after implantation. The tissue sections were incubated with antibodies against indicated antibodies (PTK7, p-Smad2/3, Id1, Ki-67). Diaminobenzidine was used as a chromogen, followed by counterstaining with hematoxylin. Incidence of apoptosis was determined with TUNEL staining. Bar, 50 μm. Ki-67 and TUNEL, Scr versus shPTK7; P < .05.