Brain Tumor Res Treat.  2020 Apr;8(1):43-52. 10.14791/btrt.2020.8.e1.

Inhibition of the Spectraplakin Protein Microtubule ActinCrosslinking Factor 1 Sensitizes Glioblastomas to Radiation

• Bonner K
• Borlay D
• Kutten O
• Quick QA

• KMID: 2500121
• DOI: http://doi.org/10.14791/btrt.2020.8.e1

Abstract

Background
: Microtubule actin crosslinking factor 1 (MACF1) is a spectraplakin cytoskeletal crosslinking protein whose function and role in cancer biology has lacked investigation. Recent studies have identified MACF1 as a novel target in glioblastomas expressed in tissue from tumor patient explants but not normal brain tissue and when silenced has an antitumorigenic impact on these tumors. Radiation as a single agent therapy to treat glioblastomas has been used for decades and has done little to improve survival of individuals diagnosed with this disease. However, contemporary clinical radiotherapy protocols have provided evidence that combinatorial radiotherapy approaches confer a therapeutic benefit in glioblastoma patients. In this study MACF1 was investigated as a radiosensitization target in glioblastomas.
Methods
: To provide context of MACF1 in glioblastomas, The Cancer Genome Atlas expression analyses were performed in conjunction with genes associated with glioblastoma evolution, while a genetic inhibitory approach, cell migratory assays, and immunofluorescence procedures were used to evaluate responses to MACF1 suppression with radiation. Additionally, expression analyses were conducted to assess co-expression of mTOR signaling pathway regulators and MACF1 in glioblastoma patient samples.
Results
: Our amalgamation approach demonstrated that negative regulation of MACF1, which was positively correlated with epidermal growth factor receptor and p70s6k expression, enhanced the sensitivity of glioblastoma cells to radiation as a consequence of reducing glioblastoma cell viability and migration. Mechanistically, the antitumorigenic effects on glioblastoma cell behaviors after radiation and impairing MACF1 function were associated with decreased expression of ribosomal protein S6, a downstream effector of p70s6k.
Conclusion
: MACF1 represents a diagnostic marker with target specificity in glioblastomas that can enhance the efficacy of radiation while minimizing normal tissue toxicity. This approach could potentially expand combinatorial radiation strategies for glioblastoma treatments via impairment of translational regulatory processes that contribute to poor patient survival.

Keyword

MACF1; Glioblastoma; Radiation; Ribosomal protein S6

Figure

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  Fig. 1 Correlative expression of MACF1 to patient survival and glioblastoma associated genes. Comparative survival curve analysis of glioblastoma patients with low MACF1 expression (<1.048; n=74) and high MACF1 mRNA expression (>1.048; n=83). MACF1 co-expression with EGFR, PTEN, p53, and CDKN2A.

• 

  Fig. 2 Down-regulation of MACF1 in genetically silenced glioblastoma cells. U251 glioblastoma cells treated with non-targeting shRNAs (A–C) and shRNAs targeting MACF1 (D–F); A172 glioblastoma cells treated with non-targeting shRNAs (G–I) and shRNAs targeting MACF1 (J–L); MACF1 expression (green); DAPI (blue); cytoplasmic MACF1 expression (arrows). Displayed are immunofluorescent images representative of three separate independent experiments with comparable results. Image J was used to measure the fluorescence intensity from a total of 50 individual cells from three separate independent experiments (n=3) and averaged. Student's t-test was used to determine statistical significance (**p<0.01; error bars: standard error of the means). Scale bar=100 µm; total magnification ×200; RFU: relative fluorescence units.

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  Fig. 3 MACF1 inhibition sensitizes glioblastoma cells to radiation. Impairing MACF1 function reduces cell viability in non-irradiated cells and enhances the efficacy of radiation. Data displayed is of a single experiment performed in duplicate and equivalent to three separate independent experiments performed in duplicate with comparable outcomes. ANOVA analysis followed by Tukey's post-hoc analysis revealed statistical significant differences between means of experimental conditions (*p<0.05; **p<0.01; error bars: standard error of the means).

• 

  Fig. 4 Radiation and suppression of MACF1 impairs glioblastoma cell migration. U251 (A–D) and A172 (E–H) cells treated with shMACF1 and irradiated with a single 5 Gy dose. Displayed are stained migrating cells in Boyden chambers and data from a single experiment performed in duplicate representative of at least three separate independent experiments performed in duplicate with comparable results 24 hours after radiation exposure. ANOVA analysis followed by Tukey's post-hoc analysis revealed statistical significant differences between means of experimental conditions (*p<0.05; **p<0.01; error bars: standard error of the means). Scale bar=200 µm; stained with crystal violet; total magnification ×100.

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  Fig. 5 Evaluation of translational regulatory proteins as mechanistic components of the radiosensitization effects of downregulating MACF1. A, B: Positive correlative expression of MACF1 with ribosomal protein S6 regulators MTOR and RPS6KB1 in clinical glioblastoma patients. C: Silencing MACF1 combined with radiation decreases p-ribosomal protein S6 expression. Immunofluorescence images displayed are from a single experiment in U251 glioblastoma cells at 24 hours and representative of three separate experiments that showed comparable results. Image J was used to measure the fluorescence intensity from a total of 50 individual cells from three separate independent experiments (n=3) and averaged. ANOVA analysis followed by Tukey's post-hoc analysis revealed statistical significant differences between means of experimental conditions (****p<0.0001; error bars: standard error of the means). Scale bar=80 µm; total magnification ×400. RFU: relative fluorescence units.

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