Epigenetic up-regulation of ribosome biogenesis and more aggressive phenotype triggered by the lack of the histone demethylase JHDM1B in mammary epithelial cells

The alterations of ribosome biogenesis and protein synthesis play a direct role in the development of tumors. The accessibility and transcription of ribosomal genes is controlled at several levels, with their epigenetic regulation being one of the most important. Here we explored the JmjC domain-containing histone demethylase 1B (JHDM1B) function in the epigenetic control of rDNA transcription. Since JHDM1B is a negative regulator of gene transcription, we focused on the effects induced by JHDM1B knock-down (KD). We studied the consequences of stable inducible JHDM1B silencing in cell lines derived from transformed and untransformed mammary epithelial cells. In these cellular models, prolonged JHDM1B downregulation triggered a surge of 45S pre-rRNA transcription and processing, associated with a re-modulation of the H3K36me2 levels at rDNA loci and with changes in DNA methylation of specific CpG sites in rDNA genes. We also found that after JHDM1B KD, cells showed a higher ribosome content: which were engaged in mRNA translation. JHDM1B KD and the consequent stimulation of ribosomes biogenesis conferred more aggressive features to the tested cellular models, which acquired a greater clonogenic, staminal and invasive potential. Taken together, these data indicate that the reduction of JHDM1B leads to a more aggressive cellular phenotype in mammary gland cells, by virtue of its negative regulatory activity on ribosome biogenesis.

5 × 10 4 cells pre-treated with TRC or left untreated were seeded in six-well plates and cultured for 10 days in control or TRC conditions. Every 3 days the cells were harvested, counted (after Trypan blue staining to exclude dead cells from the count) in a Burker Haemocytometer, and seeded again in new plates in a 1:4 (MCF 10A derived cells) or 1:5 (MDA-MB-231 derived cells) ratio. Between different points of the growth curves the doubling times (DTs) were calculated using the following formula: DT = [h × ln (a / b)] / ln (2), where h is the growth time in hours, a is the number of seeded cells, and b is the number of cells at the end of the growth time. The DTs were then used to indirectly assess the real cell number by applying the following formula: cell number = 2 (h/DT) .

Cell invasion assay
Invasion assays were performed in blind well chambers (Neuroprobe Inc.) according to the manufacturer's instructions, using 13mm-diameter polycarbonate filters (Neuroprobe Inc.) with pore size 8 μm. 5 × 10 4 cells, pre-treated with TRC or left untreated, were seeded in the upper compartment in low FBS cell culture medium, [1% and 2% respectively for MDA-MB-231 sh(1 or 2)-JHDM1B and MCF 10A sh(1 or 2)-JHDM1B], while 10% and 20% FBS (respectively) in cell culture medium were placed in the lower compartment. After a 24 h incubation at 37°C, 5% CO 2 , filters were collected and washed with water, while cells were fixed in absolute ethanol for 1 min. Lastly, cells were stained with Giemsa stain (1:10 in water) at RT for 10 min and filters were washed again twice with water. The non-invading cells were scraped off with a cotton swab. Cells were visualized with a Leitz Diaplan light microscope (Wetzlar Germany) equipped with a video camera (JVC, 3CCD, KY-F55B, Jokohama, Japan) at 10× of megnification; 5 random fields for each filter were photographed and counted.

Clonogenic assays
In a single 6-well plate, 150 cells or 500 cells, for MDA-MB-231 sh1-JHDM1B and MCF 10A sh1-JHDM1B respectively, were seeded and treated daily with TRC or left untreated. The colony number was evaluated 10-12 days later, after overnight fixation in 4% formalin at 4°C and staining with a 0.5% crystal violet solution in 25% methanol for 30 min. Cells were then washed 3 times in PBS and counted.

Generation of mammospheres
1.2 × 10 4 cells were seeded in ultra-low attachment 6-well plates and cultured in Mammary Epithelial Cell Growth Medium (MEGM, Bullet Kit, Lonza). Spheres started forming after 4-6 days and MS were counted between days 7 and 8 under an inverted microscope at 10× magnification.

MDA-MB-231 sh1-JHDM1B xenografts
2 × 10 6 MDA-MB-231 sh1-JHDM1B cells, pretreated with TRC or left untreated for 6 days, were suspended in 100 μl of PBS and injected subcutaneously into both flanks of anesthetized 5-week-old female Balb/ COlaHsd-Foxn1nu mice (Harlan Laboratories Inc.). Five mice for each of the 2 groups (pre-treated with TRC or left untreated) were injected, for a total of 10 xenografts in control conditions and 10 xenografts in JHDM1B KD condition. Mice of the two groups were watered with 3% sucrose in water or 3% sucrose supplemented with TRC 1.5 mg/ml, respectively, starting 3 days before the xenograft. Mice were euthanized nine weeks later, and the tumor masses were excised from the flanks. Tumor diameters were evaluated using a caliber. Some of the tissues were immediately frozen in liquid nitrogen for subsequent RNA extraction by the Tri-Reagent (Ambion), following the manufacturer's specifications. The remaining tissues were embedded in 4% paraffin and used to generate five-micron sections, later processed to perform the selective nucleolar staining.
All of the animal work was approved by Bologna University's Institutional Animal Care and Use Committee in accordance with national guidelines and standards. Figure 1: Evaluation of genes expression in JHDM1B KD cells. JHDM1B KD in MDA-MB-231 sh1-JHDM1B does not cause a different gene transcription of CDKN2B (p15) and CDKN1A (P21) and genes implicated in the policomb silencing complex PRC (EZH2 and BMI1). Data were obtained by RT-PCR analysis after 6 days of TRC treactement (black filled) or in control condition (white filled) and statistical analysis performed by paired Student's T-test.