FHOD1 is upregulated in glioma cells and attenuates ferroptosis of glioma cells by targeting HSPB1 signaling

Abstract

Background: As a new type of regulatory cell death, ferroptosis has been proven to be involved in cancer pathogenesis and therapeutic response. However, the detailed roles of ferroptosis or ferroptosis-associated genes in glioma remain to be clarified.

Methods: Here, we performed the TMT/iTRAQ-Based Quantitative Proteomic Approach to identify the differentially expressed proteins between glioma specimens and adjacent tissues. Kaplan-Meier survival was used to estimate the survival values. We also explored the regulatory roles of abnormally expressed formin homology 2 domain-containing protein 1 (FHOD1) in glioma ferroptosis sensitivity.

Results: In our study, FHOD1 was identified to be the most significantly upregulated protein in glioma tissues. Multiple glioma datasets revealed that the glioma patients with low FHOD1 expression displayed favorable survival time. Functional analysis proved that the knockdown of FHOD1 inhibited cell growth and improved the cellular sensitivity to ferroptosis in glioma cells T98G and U251. Mechanically, we found the up-regulation and hypomethylation of HSPB1, a negative regulator of ferroptosis, in glioma tissues. FHOD1 knockdown could enhance the ferroptosis sensitivity of glioma cells via up-regulating the methylated heat-shock protein B (HSPB1). Overexpression of HSPB1 significantly reversed FHOD1 knockdown-mediated ferroptosis.

Conclusions: In summary, this study demonstrated that the FHOD1-HSPB1 axis exerts marked regulatory effects on ferroptosis, and might affect the prognosis and therapeutic response in glioma.

Keywords: FHOD1; HSPB1; ferroptosis; glioma; prognosis.

FIGURE 1

Identification of up‐regulated FHOD1 expression in glioma tissues. (A) Volcanic maps suggested the up‐regulated and down‐regulated molecules in glioma tissues. (B, C) GSEA analysis indicated the significant pathways regulated by the differentially expressed molecules. (D) Heat map of the top 10 altered molecules. (E) The CPTAC database confirmed up‐regulated FHOD1 in glioma tissues. (F–H) In three CGGA datasets, the glioma patients with low FHOD1 expression displayed favorable prognosis. (I) The glioma patients with high FHOD1 expression displayed high risk of recurrence.

FIGURE 2

The inhibitory effect of FHOD1 knockdown on the growth of glioma cells. (A) Western blot confirmed the down‐regulation of FHOD1 expression in Ctrl or FHOD1 shRNAs stably‐expressed T98G and U251 cells. (B) Colony formation assay indicated the inhibitory effect of FHOD1 depletion on growth of glioma cells. (C, D) Quantification of cell survival determined by colony formation assay. (E, F) CCK‐8 assay indicated the inhibitory effect of FHOD1 depletion on proliferation of glioma cells. (G) The mitochondrial morphological changes revealed by electron microscope. Error bars represented the mean ± SD from three independent experiments. **p < 0.01.

FIGURE 3

Knockdown of FHOD1 improved the ferroptosis in glioma cells. (A, B) CCK‐8 assay indicated the effects of FHOD1 knockdown on cytotoxic activity of erastin in glioma cells T98G and U251. (C) Flow cytometer indicated the cellular ROS fluorescence signal after FHOD1 knockdown. (D, E) Quantification of cellular ROS levels from (C). (F, G) The intracellular Fe2+ levels in FHOD1‐deficient T98G and U251 cells. (H, I) Ferroptosis inhibitor Fer‐1 antagonized the cytotoxic activity of erastin in FHOD1 knockdown T98G and U251 cells (J) Flow cytometer indicated the cellular ROS fluorescence signal after FHOD1 knockdown and Fer‐1 treatment. (K, L) Quantification of cellular ROS levels from (J). (M, N) The intracellular Fe2+ levels in FHOD1‐deficient T98G and U251 cells treated with Fer‐1. Error bars represented the mean ± SD from three independent experiments. **p < 0.01.

FIGURE 4

FHOD1 knockdown inhibited the ferroptosis‐associated HSPB1. (A) Venn diagrams identified the significantly upregulated HSPB1. (B) The correlation between FHOD1 expression and HSPB1 expression in GBM and LGG tissues. (C, D) Western blot and qRT‐PCR confirmed the down‐regulation of HSPB1 expression in FHOD1‐depleted T98G and U251 cells. (E) After FHOD1 knockdown and Flag‐HSPB1 overexpression, the total protein was extracted and analyzed by western blot with the indicated antibodies. (F) FHOD1‐depleted T98G and U251 cells reconstituted with Flag‐HSPB1 were used to detect the cell growth rates. (G, H) Quantification of cell growth determined by colony formation assay from (F). (I, J) FHOD1‐depleted T98G and U251 cells reconstituted with Flag‐HSPB1 were used to detect the cell proliferation rates. Error bars represented the mean ± SD from three independent experiments. **p < 0.01.

FIGURE 5

The ferroptosis resistance‐induced by FHOD1 was dependent on HSPB1. (A) After FHOD1 knockdown and Flag‐HSPB1 overexpression, the total protein was extracted and analyzed by western blot with the indicated antibodies. (B, C) CCK‐8 assay indicated the cytotoxic activity of erastin in T98G and U251 cells with FHOD1 knockdown and Flag‐HSPB1 overexpression. (D) Flow cytometer indicated the cellular ROS fluorescence signal after FHOD1 knockdown and Flag‐HSPB1 overexpression. (E, F) Quantification of cellular ROS levels from (D). (G, H) The intracellular Fe2+ levels in T98G and U251 cells with FHOD1 knockdown and Flag‐HSPB1 overexpression. Error bars represented the mean ± SD from three independent experiments. **p < 0.01.

FIGURE 6

HSPB1 is hypomethylated in glioma cells. (A) UALCAN database indicated the up‐regulated HSPB1 mRNA levels in glioma tissues. (B) UALCAN database indicated the hypomethylated HSPB1 in glioma tissues. (C) The patients with hypermethylated HSPB1 displayed favorable prognosis. (D) Two CpG islands in HSPB1 gene promotor were identified by MethPrimer. (E) Bisulfite sequencing PCR was used to confirmed the methylated islands of HSPB1 promotor after FHOD1 knockdown.

FIGURE 7

The clinical significance of FHOD1 in glioma. (A) The representative immunohistochemical staining of FHOD1 on glioma tissue microarray (n = 145). Scale bars are indicated. (B, C) Protein levels of FHOD1 was quantified in glioma specimens with different grades and stages. (D) The glioma patients with low FHOD1 expression displayed favorable OS. (E) The glioma patients with low FHOD1 expression displayed favorable PFS. (F) The representative immunohistochemical staining of FHOD1 on Xiangya glioma cohorts (n = 50). Scale bars are indicated. (G) Correlation analysis of FHOD1 and HSPB1 in glioma samples. Statistical analyses were performed with the χ² test. The Pearson r indicates correlation coefficient. (H) The tumor tissues were harvested at the end of the experiment in each group. (I, J) The tumor weight and tumor volume of each group in glioma xenograft models. The asterisks (**) indicate the following: FHOD1 knockdown vs. control (p < 0.01) and FHOD1 knockdown + HSPB1 overexpression vs. FHOD1 knockdown (p < 0.01). (K) The body weight curves of each group in glioma xenograft models.