Coordinated regulation of IGF1R by HIF1α and HIF2α enhances chemoresistance in glioblastoma

Abstract

Background: This study investigates whether Hypoxia-Inducible Factor 1 alpha (HIF1α) and Hypoxia-Inducible Factor 2 alpha (HIF2α) coordinately regulate insulin-like growth factor 1 receptor (IGF1R) expression, thereby influencing chemosensitivity in glioblastoma multiforme (GBM).

Methods: We analyzed the expression and correlation of HIF1α, HIF2α, and IGF1R in glioma using The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) databases. Immunohistochemistry (IHC) was performed to detect the expression of HIF1α, HIF2α, and IGF1R in GBM tissues and cells, as well as oxygen tension. Cell cycle analysis, apoptosis assays, lactate dehydrogenase (LDH) release measurements, Western blotting, and xenograft tumor models were employed to explore the synergistic regulation of IGF1R by HIF1α and HIF2α, focusing on activation of the PI3K/AKT signaling pathway and its contribution to GBM drug resistance. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) and dual-luciferase reporter assays were used to investigate the binding sites of HIF1α and HIF2α involved in regulating IGF1R.

Results: Our study demonstrated that HIF1α and HIF2α were highly expressed in GBM tissues and hypoxia-cultured cells, and their expression positively correlated with IGF1R expression. Simultaneous knockout of HIF1α and HIF2α in GBM cells resulted in the highest LDH release and apoptosis rates under hypoxic conditions, accompanied by the most significant decrease in IGF1R, p-PDK1, and p-AKT levels. Knockout of IGF1R in tumor cells under hypoxia led to an increas of LDH release and apoptosis rates, and reduced phosphorylation of PDK1 and AKT. In addition, we demonstrated that HIF1α and HIF2α promoted IGF1R expression by binding to a specific hypoxia response element (HRE) sequence (5'-GAACGTGCCT-3') within the IGF1R promoter using dual-luciferase reporter system.

Conclusion: Glioblastoma cells, residing within a hypoxic microenvironment, exhibit high expression of HIF1α and HIF2α. These transcription factors promote the upregulation of IGF1R, which subsequently activates the PI3K/AKT signaling pathway. This activation, in turn, promotes cell proliferation and chemoresistance, ultimately contributing to tumor malignancy.

Keywords: HIF1α; HIF2α; IGF1R; chemoresistance; glioblastoma multiforme; hypoxic microenvironment; temozolomide.

FIGURE 1

(A–C) Analysis of the CGGA database reveal that Hypoxia-Inducible Factor 1α (HIF1α) and Hypoxia-Inducible Factor 2α (HIF2α) are highly expressed in glioblastoma, with HIF1α expression positively correlated with glioma grade and negatively correlation with patient survival time. (D) Immunohistochemical staining demonstrated high expression levels of HIF1α and HIF2α in clinical GBM tissues and in tumor tissues from mice following intracranial tumor implantation. Hypoxia probe detection indicated low oxygen partial pressure within the intracranial tumor-bearing tissues of mice. (E) Immunofluorescence staining and hypoxia probe detection after culturing U87 cells in 21% O₂ or 1% O₂ for 72 h showed that the oxygen partial pressure in cells cultured in 1% O₂ was significantly lower than that in the control group, and the expression levels of HIF1α and HIF2α were significantly higher than those in the control group.

FIGURE 2

(A) Western blot analysis confirmed the successful knockout of HIF1α and HIF2α in U87 and U251 cells. It also demonstrated that HIF2α expression increased after HIF1α knockout, and vice versa, HIF1α expression increased after HIF2α knockout under hypoxic conditions. (B) Cell cycle analysis showed no significant changes in the cell cycle after single knockout of either HIF1α or HIF2α; however, simultaneous knockout significantly decreased the proportion of cells in the G₁ phase and significantly increased the proportion in the G₂/M + S phase. (C, D) Lactate Dehydrogenase (LDH) and apoptosis assays showed that both LDH release and apoptosis rates were reduced in the hypoxia group compared to the normoxia group. Single knockout of either HIF1α or HIF2α increased LDH release and apoptosis rates, while simultaneous knockout resulted in the highest LDH release and apoptosis rates. (E–G) Following intracranial orthotopic implantation of control, HIF1α-ko, HIF2α-ko, or HIF1α/HIF2α-ko cells (8 × 10⁴) and treatment with the same dose of Temozolomide (TMZ), tumor volume and weight were reduced in the HIF1α or HIF2α single knockout groups. The smallest tumor volume and weight were observed in the HIF1α/HIF2α double knockout group. (H) With the same dose of TMZ treatment, the survival time of mice in the HIF1α or HIF2α single knockout groups was prolonged compared to the control group, and the survival time of mice in the HIF1α and HIF2α double knockout group was the longest. *P < 0.05 represents the difference between HIF2α/HIF2α-ko and vector control under hypoxia, **P < 0.01 represents the difference between normoxia and hypoxia-treated groups, ^# P > 0.05 represents the difference between vector and single HIF1α or HIF2α knockout under hypoxia, and all comparisons were determined using one-way ANOVA.

FIGURE 3

(A) Immunohistochemical staining showed high expression levels of Insulin-like Growth Factor 1 Receptor (IGF1R) in tumor tissues. (B) Immunofluorescence staining revealed low IGF1R expression in GBM cells under normoxic conditions, but significantly increased expression after 72 h of culture in a 1% O₂ environment. (C–E) TCGA and CGGA database analyses showing a positive correlation between HIF1α,HIF2α,and IGF1R expression. (F) RT-qPCR analysis showed that IGF1R expression decreased after single knockout of either HIF1α or HIF2α; the most significant decrease in IGF1R expression was observed after simultaneous knockout of HIF1α and HIF2α. (G) Western blot analysis showed increased expression of IGF1R, p-PDK1, p-AKT, and mTOR after hypoxic culture. Single knockout of either HIF1α or HIF2α reduced the expression of IGF1R, p-PDK1, p-AKT, and mTOR, while simultaneous knockout of HIF1α and HIF2α resulted in the lowest expression levels of IGF1R, p-PDK1, p-AKT, and mTOR. *P < 0.05 represents the difference between normoxia and hypoxia-treated groups, **P < 0.05 represents the difference between vector and single HIF1α or HIF2α knockout under hypoxia, and ***P < 0.01 represents the difference between vector and both HIF1α and HIF2α knockout under hypoxia. All comparisons were determined using Student’s t-test.

FIGURE 4

(A) Western blot analysis confirmed the successful knockout of IGF1R in U87 and U251 cells, and that IGF1R knockout led to decreased expression of p-PDK1, p-AKT, and mTOR. (B, C) LDH and apoptosis assays showed that IGF1R knockout increased cellular LDH release and apoptosis rates. *P < 0.05, determined using Student’s t-test.

FIGURE 5

(A) Chromatin Immunoprecipitation-qPCR (ChIP-qPCR) experiments showed that HIF1α and HIF2α bind to the hypoxia response element (HRE) region (−1,253 to −1,262) of the IGF1R promoter and promote transcription. (B) IGF1R promoter activity was increased under hypoxic culture compared to normoxic culture, and the promoter activity significantly decreased after mutation of the binding site. (C) IGF1R promoter activity was lower in cells with single knockout of either HIF1α or HIF2α compared to control cells, and the lowest promoter activity was observed in cells with simultaneous knockout of HIF1α and HIF2α. (D) Under hypoxic conditions (1% O₂), HIF1α and HIF2α promote high expression of IGF1R in cells. The highly expressed IGF1R activates the PI3K/AKT signaling pathway, thereby promoting cell proliferation and chemoresistance. *P < 0.05, determined using one-way ANOVA or Student’s t-test.