Role of KDM2B epigenetic factor in regulating calcium signaling in prostate cancer cells

Abstract

KDM2B, a histone lysine demethylase, is expressed in a plethora of cancers. Earlier studies from our group, have showcased that overexpression of KDM2B in the human prostate cancer cell line DU-145 is associated with cell adhesion, actin reorganization, and improved cancer cell migration. In addition, we have previously examined changes of cytosolic Ca²⁺, regulated by the pore-forming proteins ORAI and the Ca²⁺ sensing stromal interaction molecules (STIM), via store-operated Ca²⁺ entry (SOCE) in wild-type DU-145. This study sought to evaluate the impact of KDM2B overexpression on the expression of key molecules (SGK1, Nhe1, Orai1, Stim1) and SOCE. Furthermore, this is the first study to evaluate KDM2B expression in circulating tumor cells (CTCs) from patients with prostate cancer. mRNA levels for SGK1, Nhe1, Orai1, and Stim1 were quantified by RT-PCR. Calcium signals were measured in KDM2B-overexpressing DU-145 cells, loaded with Fura-2. Blood samples from 22 prostate cancer cases were scrutinized for KDM2B expression using immunofluorescence staining and the VyCAP system. KDM2B overexpression in DU-145 cells increased Orai1, Stim1, and Nhe1 mRNA levels and significantly decreased Ca²⁺ release. KDM2B expression was examined in 22 prostate cancer patients. CTCs were identified in 45 % of these patients. 80 % of the cytokeratin (CK)-positive patients and 63 % of the total examined CTCs exhibited the (CK + KDM2B + CD45-) phenotype. To conclude, this study is the first to report increased expression of KDM2B in CTCs from patients with prostate cancer, bridging in vitro and preclinical assessments on the potentially crucial role of KDM2B on migration, invasiveness, and ultimately metastasis in prostate cancer.

Keywords: Calcium signaling; Circulating tumor cells; KDM2B; Orai1; Prostate cancer; Stim1; Store-operated Ca2+ entry.

Fig. 1

Effect of KDM2B overexpression on SGK1, Orai1, Stim1, and Nhe1 transcription in the prostate cancer cell line DU-145. mRNA expression of SGK1 (A), Orai1 (B), Stim1 (C), and Nhe1 (D) was measured by RT-PCR in DU-145 cells overexpressing KDM2B compared to wild-type cells. Data are presented as ratios of the respective genes with β-actin, with control cells arbitrarily set to 1. Data are shown as mean ± SD and n = 3 represents the independent experiments. **p < 0.01 signifies a difference that is statistically significant (unpaired Student t-test). n.s.: non-statistically significant.

Fig. 2

KDM2B overexpression’s effect on Ca²⁺ release and SOCE in the prostate cancer cell line DU-145. A) Representative trace of Fura-2 fluorescence ratio following extracellular Ca²⁺ removal, add-on of thapsigargin (line and arrow above trace), and re-introduction of extracellular Ca²⁺ in DU-145 overexpressing KDM2B (black circles) compared to wild-type cells (white circles). Arithmetic means (±SEM, n = 3) of peak (B) and slope (C) elevation in Ca²⁺ from intracellular stores after addition of thapsigargin (1 μM) and of peak (D) and slope (E) increase of SOCE after re-addition of extracellular Ca2⁺ in DU-145 cells overexpressing KDM2B (black bars) compared to wild-type cells (white bars). **p < 0.01 signifies a difference that is statistically significant.

Fig. 3

Expression of KDM2B in prostate cancer patients’ blood samples. A) Proportion of cytokeratin CTCs-positive patients with (CK+/KDM2B+/CD45−) and (CK+/KDM2B−/CD45−) phenotypes. Β) Mean proportions of overall CTCs exhibiting the phenotypes (CK+/KDM2B+/CD45−) and (CK+/KDM2B−/CD45−). C) Representative photos of patient’s CTCs expressing CK (red), an epithelial CTC marker, KDM2B (green), the biomarker of interest, and CD45 (purple), a leukocyte marker, in samples isolated from prostate cancer patients. DAPI was used for staining the nuclei (blue). Magnification 20×. Scale bar = 10 μm.

Supplementary Fig. 1

KDM2B expression in spiked controls to evaluate the method and simulate the CTC microenvironment. Expressed CK (red), an epithelial CTC marker, KDM2B (green), the biomarker of interest, and CD45 (purple), a leukocyte marker, in H1299 spiked controls made by combining 100,000 PBMCs with 1000 H1299 cancer cells. DAPI was used for staining the nuclei (blue). Magnifications 20× and 40×. Scale bar = 10 μm.