CDKN2A (p16INK4A) affects the anti‑tumor effect of CDK inhibitor in somatotroph adenomas

Abstract

The altered cell cycle is associated with aberrant growth factor signaling in somatotroph adenoma, which is the primary cause of acromegaly. The aim of the present study was to investigate the pathological role of the INK4 family and evaluate the effectiveness of CDK4 inhibitor, palbociclib, in somatotroph adenoma. RNA‑Seq, RT‑PCR, and immunohistochemistry were applied to measure the levels and correlations of the INK4 family with angiogenesis, CDKs, EMT, and therapeutic targets. MTS, flow cytometry, and ELISA were used to investigate the bio‑activity in GH3 and GT1‑1 cell lines after palbociclib treatment. Compared with lactotroph adenoma, gonadotroph adenoma, and corticotroph adenoma, somatotroph samples demonstrated higher expression of CDKN2A and SSTR2 but a lower expression of EGFR, CDK4, and CDH2 (P<0.05). CDKN2A positively correlates with SSTR2, and negatively with CDK4, EGFR, and CDH2. Patients with lower CDKN2A had larger tumor size (P=0.016) and more invasive potential (P=0.023). Palbociclib inhibited cell proliferation, induced G1 phase arrest, reduced GH/IGF‑1 secretion of GH3 and GT1‑1 cell lines (P<0.05), and had a more prominent role in GH3 cells (P<0.05). CDKN2A inhibited the bio‑activity by modulating CDK4, and high CDKN2A predicted the insensitivity to CDK4 inhibitor, palbociclib, in somatotroph adenoma patients. In summary, the present study shows CDKN2A inhibited the bio‑activity by modulating CDK4, and high CDKN2A predicts the insensitivity to CDK4 inhibitor, Palbociclib, in somatotroph adenoma patients.

Keywords: somatotroph adenomas; INK4 family; CDKN2A (p16INK4A); palbociclib; CDK4.

Figure 1

Correlation analyzes of INK4 family with tumor functions by 24 SOMA RNA-Seq TPM data. (A) Angiogenesis. The most negative gene was EGFR. (B) CDKs family. The most negative gene was CDK4, and the most positive gene was CDK5. (C) EMT. The most negative gene was CDH2, and the most positive was SNAI1. (D) Therapeutic targets of SOMA. The most negative gene was GHR, the most positive gene was SSTR2.

Figure 2

The characteristic profile of the INK4 family in pituitary adenoma. (A) Correlation analysis among INK4 family with the characteristic molecule related to tumor behavior. (B) The mRNA level of INK4 family and characteristic molecule related to tumor behavior in subtypes of pituitary adenomas. ^*P<0.05, ^**P<0.01, ^***P<0.001, n=8.

Figure 3

Immunohistochemistry of CDKN2A, CDH2, CDK4, EGFR and SSTR2 in pituitary and pituitary adenomas. Magnification, ×100; bar=100 µm.

Figure 4

The bio-activity effect of CDK4/6 inhibitor, Palbociclib, of GH3 and GT1-1 cell line in various concentrations (1, 5, and 20µM). (A and D) The inhibition induced by Palbociclib occurred in a time- and dose-dependent manner, whether GH3 or GT1-1 cells. ^*P<0.05, ^**P<0.01, ^***P<0.001, n=8. (B and E) Palbociclib inhibited the secretion of GH/IGF-1 in GH3 and GT1-1 cell lines. (C and F) Palbociclib induced the G1 phase arrest in GH3 and GT1-1 cell lines. (G) Statistical analysis of the change of bio-activity after 72 h treatment between GH3 and GT1-1 cell lines, compared to the control group (%). ^*P<0.05, ^**P<0.01, ^***P<0.001, n=3.

Figure 5

Palbociclib relieved the levels of CDH2, CDK4 and EGFR accompanied by upregulation of CDKN2A in various concentrations (1, 5, and 20 µM). (A) Bands of western blot assay. (B) Statistical analysis of mRNA level in RT-qPCR assay. compared to control group. ^*P<0.05, ^**P<0.01, ^***P<0.001, n=3.