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. 2023 Aug 30;24(17):13467.
doi: 10.3390/ijms241713467.

Pyk2/FAK Signaling Is Upregulated in Recurrent Glioblastoma Tumors in a C57BL/6/GL261 Glioma Implantation Model

Jescelica Ortiz Rivera  1 , Grace Velez Crespo  1 , Mikhail Inyushin  1 , Yuriy Kucheryavykh  1 , Lilia Kucheryavykh  1
Affiliations

Pyk2/FAK Signaling Is Upregulated in Recurrent Glioblastoma Tumors in a C57BL/6/GL261 Glioma Implantation Model

Jescelica Ortiz Rivera et al. Int J Mol Sci. .

Abstract

The majority of glioblastomas (GBMs) recur shortly after tumor resection and recurrent tumors differ significantly from newly diagnosed GBMs, phenotypically and genetically. In this study, using a Gl261-C57Bl/6 mouse glioma implantation model, we identified significant upregulation of proline-rich tyrosine kinase Pyk2 and focal adhesion kinase (FAK) phosphorylation levels-pPyk2 (579/580) and pFAK (925)-without significant modifications in total Pyk2 and FAK protein expression in tumors regrown after surgical resection, compared with primary implanted tumors. Previously, we demonstrated that Pyk2 and FAK are involved in the regulation of tumor cell invasion and proliferation and are associated with reduced overall survival. We hypothesized that the use of inhibitors of Pyk2/FAK in the postsurgical period may reduce the growth of recurrent tumors. Using Western blot analysis and confocal immunofluorescence approaches, we demonstrated upregulation of Cyclin D1 and the Ki67 proliferation index in tumors regrown after resection, compared with primary implanted tumors. Treatment with Pyk2/FAK inhibitor PF-562271, administered through oral gavage at 50 mg/kg daily for two weeks beginning 2 days before tumor resection, reversed Pyk2/FAK signaling upregulation in recurrent tumors, reduced tumor volume, and increased animal survival. In conclusion, the use of Pyk2/FAK inhibitors can contribute to a delay in GBM tumor regrowth after surgical resection.

Keywords: FAK; Pyk2; glioblastoma; recurrent tumor.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Pyk2 and FAK signaling is upregulated in tumors regrown after surgical resection, compared with primary implanted tumors in GL261-C57BL/6 mouse glioma implantation model. Representative Western blots and quantifications with individual values, for relative levels of expression of total and phosphorylated Pyk2 and FAK in primary implanted and regrown-after-resection tumors are shown. Tumors were analyzed 14 days after primary implantation (for primary implanted tumors) and 14 days after tumor resection (for regrown-after-resection tumors). Relative values in regrown tumors compared with primary implanted tumors are shown. Actin was used as a loading control. Mean ± S.E. and significant differences from the control (*) (p < 0.005). N = 5.
Figure 2
Figure 2
Probability of survival in glioblastoma patients depends on Pyk2 (PTK2B) and FAK (PTK2) gene expression. Kaplan–Meier survival analysis for Pyk2 (A) and FAK (B) gene expression level is shown. Microarray data analysis of 619 patients from the Cancer Genome Atlas (TCGA, Glioblastoma) were used. Curve comparison was performed with use of log-rank (Mantel-Cox) test (p = 0.009 for (A) and p = 0.0275 for (B)). Cut-off values were calculated by receiver operating characteristic (ROC) analysis.
Figure 3
Figure 3
PF-562271 reduces Pyk2 and FAK phosphorylation in tumors regrown after surgical resection in a GL261-C57BL/6 mouse glioma implantation model. (A) Schematic representation of experimental procedures. (B) Representative Western blots and quantifications with individual values of relative expression of total and phosphorylated Pyk2 and FAK, together with Iba1 expression. The displayed values represent the relative differences in the PF-562271 treatment group compared to the vehicle group. (C) Immunofluorescence confocal images of tumors and (D) quantification of galactose-binding lectin-stained cells in tumors regrown after surgical resection, with and without PF-562271 treatment. PF-562271 (50 mg/kg/day) was provided orally for 14 consecutive days after resection surgery, beginning 2 days before the tumor resection procedure. Actin was used as a loading control. Scale bar is 100 µm. Mean ± S.E. and significant differences from the control (*) (p < 0.005). N = 5.
Figure 4
Figure 4
PF-562271 treatment downregulates Cyclin D1 expression and the Ki67 proliferation index in tumors regrown after surgical resection in a GL261-C57BL/6 mouse glioma implantation model. (A) Representative Western blots and quantifications with individual values for the relative level of Cyclin D1 protein expression in the tumors of animals that received vehicle or PF-562271 for 14 days after tumor resection. The displayed values represent the relative differences in the PF-562271 treatment group compared to the vehicle group. Actin was used as a loading control. (B) Representative immunofluorescence confocal images, and (C) a quantification of cells expressing the Ki67 marker within regrown tumors following surgical resection. The tumors were treated with PF-562271 (50 mg/kg/day) or vehicle for a continuous 14-day duration post-resection, beginning 2 days prior to the tumor removal procedure. Arrows indicate cells expressing Ki67. Scale bar is 100 µm. Mean ± S.E. and significant differences from the control (*) (p < 0.005). N = 5.
Figure 5
Figure 5
PF-562271 provided 2 days before the tumor resection and, then, during the post-surgical period reduced the growth of recurrent tumors and increased survival rates in a GL261-C57BL/6 mouse glioma implantation model. (A) Hematoxylin and eosin staining of brain sections, encompassing entire tumors, and (B) quantification with individual values of tumor size. The tumor edge is delineated by a dotted outline. The tumor resection was performed 14 days after tumor implantation. PF-562271 or vehicle were given to animals 2 days before and, then, after the tumor resection, 50 mg/kg/day, every day. Data are presented for tumors regrown 14 days after the surgical resection. Tumor size evaluation was performed immediately after the treatment course termination. (C) Kaplan–Meier estimates of overall survival probability for animals treated with vehicle and with PF-562271 beginning 2 days before the tumor resection and continuing during the entire post-surgical period until animal mortality. Curve comparison was performed using the log-rank (Mantel–Cox) test (p < 0.05). The results are presented as the mean ± S.D. with significant differences from the vehicle group (*) (p < 0.05). Scale bar: 100 µm. N = 5.
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