Int J Stem Cells.  2023 Aug;16(3):315-325. 10.15283/ijsc22097.

Parathyroid Hormone-Related Protein Promotes the Proliferation of Patient-Derived Glioblastoma Stem Cells via Activating cAMP/PKA Signaling Pathway

• Guo Z
• Huang T
• Liu Y
• Liu C

• KMID: 2545231
• DOI: http://doi.org/10.15283/ijsc22097

Abstract

Background and Objectives
Glioblastoma (GBM) is an aggressive primary brain tumor characterized by its hetero-geneity and high recurrence and lethality rates. Glioblastoma stem cells (GSCs) play a crucial role in therapy resistance and tumor recurrence. Therefore, targeting GSCs is a key objective in developing effective treatments for GBM. The role of Parathyroid hormone-related peptide (PTHrP) in GBM and its impact on GSCs remains unclear. This study aimed to investigate the effect of PTHrP on GSCs and its potential as a therapeutic target for GBM.
Methods and Results
Using the Cancer Genome Atlas (TCGA) database, we found higher expression of PTHrP in GBM, which correlated inversely with survival. GSCs were established from three human GBM samples obtained after surgical resection. Exposure to recombinant human PTHrP protein (rPTHrP) at different concentrations significantly enhanced GSCs viability. Knockdown of PTHrP using target-specific siRNA (siPTHrP) inhibited tumorsphere formation and reduced the number of BrdU-positive cells. In an orthotopic xenograft mouse model, suppression of PTHrP expression led to significant inhibition of tumor growth. The addition of rPTHrP in the growth medium counteracted the antiproliferative effect of siPTHrP. Further investigation revealed that PTHrP increased cAMP concentration and activated the PKA signaling pathway. Treatment with forskolin, an adenylyl cyclase activator, nullified the antiproliferative effect of siPTHrP.
Conclusions
Our findings demonstrate that PTHrP promotes the proliferation of patient-derived GSCs by activating the cAMP/PKA signaling pathway. These results uncover a novel role for PTHrP and suggest its potential as a therapeutic target for GBM treatment.

Keyword

Patient-derived glioblastoma stem cells; PTHrP; Proliferation; cAMP; PKA

Figure

• 

  Fig. 1 Culture and immunostaining characterization of patient-derived GSCs. (A) Comparison of PTHrP mRNA expression in samples between GBM (n=153) and LGG (n=513). ***p<0.001 versus normal tissue. (B) Kaplan–Meier survival analysis of GBM and LGG patients of lower and higher levels of PTHrP-mRNA expression. Human GSCs were isolated from three post-surgical patients (GSC-01, GSC-02, and GSC-03), and the samples’ detailed information (C) and H&E staining (D) were shown. Scale bar=50 μm. (E) After culturing for 5∼7 days, 80∼200 μm tumorspheres were observed. Scale bar=200 μm. (F) These tumorspheres were demonstrated using CD133 and nestin. (G) The single patient-derived GSCs were identified by CD133 and SOX2. The square frames are enlarged to identify the colocalization of CD133 and SOX2 staining. Scale bar=50 μm.

• 

  Fig. 2 PTHrP increases cell viability and tumorsphere formation of patient-derived GSCs. (A∼C) Three patient-derived GSCs (GSC-01, GSC-02, and GSC-03) were treated with a series of recombinant human PTHrP proteins (rPTHrP; 1, 3, 5, 10, 50, and 100 nM/ml) for 3 days, and cell viability was detected by CCK‐8 assay. *p<0.05, **p<0.01, ***p<0.001 versus control (Ctrl) group. (D) Patient-derived GSCs were transfected with FAM-labeled non-specific siRNAs (siNC) or PTHrP-targeted siRNA (siPTHrP). At the next day, more than 90% of transfected cells were observed. Scale bar=400 μm. (E, F) The expression of PTHrP was detected by western blot. Data from three independent experiments (n=3) were presented as the ratio of PTHrP to β‐Actin. ***p<0.001 versus the siNC group. After transfecting with siNC or siPTHrP, the tumorsphere number (G∼I) and the mean diameter (J∼L) were measured at 3 days, 5 days, and 7 days. Data were represented as the mean±SD of three independent experiments (n=3). (M, N) The PTHrP expression wsa examined by western blot. Data from three independent experiments (n=3) were presented as the ratio of PTHrP to β‐Actin. ***p<0.001 versus shNC group. The luciferase-expressing shNC GSCs or KD-PTHrP GSCs were used to establish the orthotopic glioma mouse model. (O, P) The in vivo imaging of shNC GSCs and KD-PTHrP GSCs in tumor-bearing mice at different time points after injection. Data are presented as the mean±standard deviation of five independent experiments (n=5). *p<0.05, **p<0.01 versus the shNC group.

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  Fig. 3 Three patient-derived GSCs (GSC-01, GSC-02 and GSC-03) were transfected for 6 hours by siNC or siPTHrP. Then, knockdown PTHrP cells were exposed with 50 nM/ml rPTHrP or equal volume of the medium. (A) After culturing for 3 days, BrdU (10 mg/ml) incorporation assay was used for detecting proliferation cells. Scale bar=100 μm. (B) The value represents the mean±standard deviation of three independent experiments (n=3). *p<0.05 versus siNC group; ###p<0.001 versus siPTHrP group.

• 

  Fig. 4 PTHrP increases cAMP concentration and promotes PKA activation. After the transfection, cells were treated with 50 nM/ml rPTHrP for 3 days. The control group just contained the same volume of the medium. The concentration of cAMP (A∼C) and the PKA (D∼F) activity were measured by immunoassay and ELISA, respectively. The value represents the mean±standard deviation of five independent experiments (n=5). *p<0.05, **p<0.01 versus siNC group; ###p<0.001 versus siPTHrP group.

• 

  Fig. 5 cAMP/PKA signaling pathway is involved in regulating the proliferation effect of PTHrP in patient-derived GSCs. Three patient-derived GSCs (GSC-01, GSC-02, and GSC-03) were transfected with siNC or siPTHrP. The siPTHrP cells were randomly allocated into two groups. One group was treated with 10 μM forskolin, the other just add an equal volume of the medium. (A) After culturing for three days, cell proliferation was analyzed using BrdU incorporation. Scale bar=100 μm. (B) The value represents the mean±standard deviation of three independent experiments (n=3). *p<0.05 versus siNC group; ###p<0.001 versus siPTHrP group. (C) The illustration depicts the mechanism that PTHrP regulates the proliferation of patient-derived GSCs. PTHrP is secreted by GSCs to the extracellular environment. A high level of PTHrP significantly increases cAMP concentration via autocrine or paracrine routes. The PKA is activated due to a high level of cAMP, eventually promoting the proliferation of patient-derived GSCs. PTHR1: parathyroid hormone 1 receptor, PTHR2: parathyroid hormone 2 receptor, AC: adenylyl cyclase.

Reference

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