Delta/notch-like epidermal growth factor-related receptor (DNER) orchestrates stemness and cancer progression in prostate cancer

Abstract

DNER, Delta/Notch-like epidermal growth factor (EGF)-related receptor, is a neuron-specific transmembrane protein carrying extracellular EGF-like repeats. The function of DNER in prostate cancer has not been evaluated. Here, we showed that the upregulation of DNER is observed in various cancers, including prostate cancer. Knockdown of DNER in PC-3 cells inhibited cell proliferation, migration and invasion as well as tumorigenesis in PC-3 xenografts. DNER knockdown specifically inhibited cell growth in spheroids. RT-PCR and western blot analysis were performed, and CD44, HES1 and GLI1 expression was significantly decreased in DNER knockdown cells. Thus, DNER promotes prostate cancer progression and the growth of PC-3 cells by modulating the primary genes of cancer stem cells.

Keywords: DNER; metastasis; proliferation; prostate cancer; stemness.

Figure 1

Expression levels of DNER between normal and cancer tissues. Expression analysis of DNER by RT-PCR in different between normal and cancer tissues from brain (A), lung (B), and pancreas (C). * P < 0.05, ** P < 0.01 versus control values. (D). Representative findings of tumors stained strongly (left) or benign tissues weakly (right). Paraffin-embedded human tissues samples were analyzed for DNER expression by IHC using anti-DNER antibody.

Figure 2

Depletion of DNER abolishes prostate cancer cell growth, migration, invasion. DNER siRNA was transfected into PC3 cells and the stable cell lines were generated by selection using puromycin and the expression of DNER was detected by RT-PCR (A). After selection the cells were plated in either anchorage dependent (liquid culture) or anchorage independent (soft agar) culture conditions. Cell growth was measured by MTT assay after 3 days (B) or alamar blue staining after one week for soft agar assay (C). (D) Cell cycle assay was used to examine the effects of DNER knockdown on cells cycle by flow cytometry analysis, and cell population (%) in each phase was quantified. (E) We transfected PC3 cells with DNER siRNA or control siRNA and detected changes in their motility using wound healing assay. The migration rate was correlated to cell migration ability. (F) Transwell assay was used to detect the effects of DNER knockdown on cells invasion. Changes in the number of cells penetrating the membrane in Transwell invasion assay. The bars represent the mean values ± SD triplicate (n = 3). * P < 0.05, ** P < 0.01 versus control values. CNTL: control group.

Figure 3

Depletion of DNER inhibits prostate cancer cell tumorigenesis. PC3 prostate cancer cells transduced with either lentiviral vectors DNER siRNA or empty vector were injected into nude mice. The ability of these cells to form tumors was monitored. The bars represent the mean values ± SD triplicate (n = 3). * P < 0.05, ** P < 0.01 versus control values. CNTL: control group.

Figure 4

Depletion of DNER depresses prostate cancer cell stemness. A. Spheroid assay in PC3 cells transduced with DNER siRNA or empty vector (control) was applied to test the features of cancer stem cell. Spheroid forming capacity is depicted in % versus CNTL set to 100%. B. The expression of CD44 by RT-PCR after DNER knockdown. The expression in spheroid assay was use as a positive control. C. qPCR data of HES1, GLI1 and HEY1 expression in PC3 cells transduced with lentiviruses carrying vectors with the DNER siRNA or empty vector (control). Expression values were normalized to HES1, GLI1 and HEY1 expression in control cells was set to 100%. D. Representative Western blots of CD44, HES1, GLI1, HEYs and β-actin of PC3 cells transduced with lentivirus carrying vectors with the DNER siRNA or from control cells transduced with lentivirus carrying an empty vector. The bars represent the mean values ± SD triplicate (n = 3). * P < 0.05, ** P < 0.01 versus control values. CNTL: control group.