Convergence of oncogenic and hormone receptor pathways promotes metastatic phenotypes

Abstract

Cyclin D1b is a splice variant of the cell cycle regulator cyclin D1 and is known to harbor divergent and highly oncogenic functions in human cancer. While cyclin D1b is induced during disease progression in many cancer types, the mechanisms underlying cyclin D1b function remain poorly understood. Herein, cell and human tumor xenograft models of prostate cancer were utilized to resolve the downstream pathways that are required for the protumorigenic functions of cyclin D1b. Specifically, cyclin D1b was found to modulate the expression of a large transcriptional network that cooperates with androgen receptor (AR) signaling to enhance tumor cell growth and invasive potential. Notably, cyclin D1b promoted AR-dependent activation of genes associated with metastatic phenotypes. Further exploration determined that transcriptional induction of SNAI2 (Slug) was essential for cyclin D1b-mediated proliferative and invasive properties, implicating Slug as a critical driver of disease progression. Importantly, cyclin D1b expression highly correlated with that of Slug in clinical samples of advanced disease. In vivo analyses provided strong evidence that Slug enhances both tumor growth and metastatic phenotypes. Collectively, these findings reveal the underpinning mechanisms behind the protumorigenic functions of cyclin D1b and demonstrate that the convergence of the cyclin D1b/AR and Slug pathways results in the activation of processes critical for the promotion of lethal tumor phenotypes.

Figure 1. Cyclin D1b induces protumorigenic phenotypes and a unique gene expression program associated with metastasis.

(A) Control (LN-vec) and cyclin D1b clones expressing low (LN-D1b [L]) and high (LN-D1b [H]) levels of cyclin D1b were hormone deprived for 72 hours. Expression of cyclin D1b protein was analyzed in the presence or absence of DHT 24 hours after treatment. (B) Control or cyclin D1b–expressing LNCaPs were plated in soft agar in androgen-proficient (FBS) or androgen-depleted (CDT) conditions and cultured for a period of 4 weeks, after which colonies greater than 75 μm in size were counted. (C) Control or cyclin D1b–expressing cells were seeded in the upper chamber of a Boyden invasion chamber and allowed to invade through the matrix toward androgen-proficient (FBS) or androgen-deprived (CDT) chemoattractants for 24 hours. Cells were fixed and DAPI stained; the total number of invading cells was reported. (D) Heat map of the differential gene expression profile regulated by cyclin D1a and cyclin D1b (cluster 1), cyclin D1a only (cluster 2), or cyclin D1b only (cluster 3). Genes shown demonstrated a false discovery rate of 1% or less and an absolute fold change of 2 or more. (E) Venn diagram comparing all cyclin D1a vs. cyclin D1b genes using a 2.0-fold cut off. Error bars represent mean ± SEM. **P < 0.01; ***P < 0.001.

Figure 2. Cyclin D1b enhances SNAI2 (Slug) expression through cooperation with the AR axis.

(A) Left: control and cyclin D1b–expressing cells were incubated with the AR inhibitor Casodex or ethanol (EtOH) control for 24 hours, RNA harvested, and relative SNAI2 levels determined. Right: control and cyclin D1b cells were cultured in androgen-proficient or androgen-depleted conditions for 72 hours and relative levels of Slug determined. Control cells cultured in androgen-proficient medium serve as a positive control. (B) Left: LNCaP and LAPC4 cells were cultured in androgen-proficient conditions and relative expression of cyclin D1b and SNAI2 levels determined. CDK4 (protein) and GAPDH (transcript) serve as controls. Right: LAPC4 cells were hormone deprived and stimulated with EtOH (0.1%), DHT (1 nM), and/or Casodex (10 μM) for 24 hours and SNAI2 expression analyzed (normalizing to GAPDH). (C) The AR-negative cell line PC3 was transfected with cyclin D1b constructs in biological duplicate, and relative levels of transcript b and SNAI2 transcript determined. ddH₂O serves as a non-template control (NTC). (D) Schematic of Morpholino mechanism of action in CCND1 alternative splicing. (E) Increasing amounts of Morpholino were introduced to LNCaP cells and relative levels of transcript b determined by qPCR after 72 hours (normalized to GAPDH). (F) LNCaP cells were treated with 12 μM Morpholino for 72 hours and levels of cyclin D1b determined. (G) Cells were treated as in E, and levels of transcript b and SNAI2 are shown. GAPDH serves as a control. Error bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3. Cyclin D1b interacts with AR on chromatin and enhances AR occupancy at SNAI2 regulatory loci.

(A) Androgen-depleted LNCaP cells were stimulated with 1 nM DHT for the indicated time points and relative expression of SNAI2 and KLK3 transcript levels determined. (B) Androgen proficient LNCaP lysates expressing a 3× flag cyclin D1b construct were fractionated into soluble and chromatin-tethered lysates and subjected to immunoprecipitation of AR or Flag. 10% input and IgG served as positive and negative controls, respectively, while GAPDH and histone H4 served as soluble and chromatin-tethered specific controls, respectfully. (C) LNCaP vector and cyclin D1b cells were androgen depleted for 72 hours and then stimulated with either DHT (10 nM) or EtOH (0.1%) for 3 hours. Samples were harvested for ChIP analysis, and percentage (input) occupancy of AR (top panel), cyclin D1b (middle), and acetylated histone H4 (bottom panel) are reported for the SNAI2 and the KLK3 loci (D). Error bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001

Figure 4. Cyclin D1b promotes chromosomal confirmations associated with active transcription in response to androgen.

(A) LNCaP cells were starved of hormone for 72 hours and treated with 10 nM DHT for 3 hours. Cells were fixed, digested with the HindIII endonuclease, and ligated; total DNA was purified. Relative distance between the constant region (proximal to AROR1 of the SNAI2 gene) and 4 test regions spanning the SNAI2 gene and downstream sequences was determined using TaqMan qPCR. Top: 100 ng of purified ligated DNA was used to test individual primer sets for each test site to ensure formation of single bands specific to ligation products. Bottom: representative images of PCR products of each ligated site in the presence or absence of DHT. A control region lacking HindIII restriction sites serves as a genomic loading control. (B) LNCaP-Vec and LNCaP-D1b cells were treated as in A, and frequency of ligation is plotted as relative to ligation frequency of parental EtOH controls, after normalizing for total DNA content (control region). (C) ChIP sequencing data of AR occupancy across the cell cycle (C. McNair and K.E. Knudsen, unpublished observations) in LNCaP cells in response to 3 hours of (10 nM) DHT. AR occupancy within the SNAI2 gene is indicated by peaks, and proximal 3C test sites are designated by triangles. Error bars represent mean ± SEM. ***P < 0.001.

Figure 5. Slug is necessary and sufficient for cyclin D1b–mediated prometastatic and tumorigenic phenotypes.

(A) Control and cyclin D1b cells were treated with a pool of siRNAs directed against the SNAI2 transcript for 72 hours and harvested for RNA (left) and protein (right). Relative levels of SNAI2 and Slug are reported. (B) siRNA-treated cells were cotransfected with GFP and allowed to invade the Boyden chamber matrix toward an androgen-proficient gradient for 24 hours, after which they were fixed and GFP-positive cells counted. Left panel shows representative fields of GFP-positive cells. Original magnification, ×10. (C) LNCaP control, Slug-expressing, and Slug- and cyclin D1b–expressing cells (left) were plated in soft agar and cultured in androgen-proficient conditions for 4 weeks. Colonies greater than 75 μm were counted for colony formation (middle). Right panel shows representative colony growth for each cell line after 4 weeks. Original magnification, ×20 (inset). (D) A polyclonal population of cells expressing Slug was probed by immunofluorescence for DAPI (panel 1), Slug (panel 2) and E-cadherin (panel 3). Merged images are represented in panel 4. Original magnification, ×40. DAPI serves as a nuclear control. Error bars represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6. Slug enhances growth of AR-positive PCa cells.

(A) Control or LN-SLUG cells were injected into the flanks of nude mice and the percentage of tumor-free mice is reported as a function of time. (B) Left: relative tumor growth of control or LN-SLUG cells is plotted as fold increase in volume relative to time of detectable tumor formation. Right: control and LN-SLUG tumors at 8 weeks after palpable tumor formation. (C) Left: Prior to sacrifice, animals were injected with BrdU for 24 hours. Tumor sections were stained for the presence of BrdU incorporation, and 3 random fields from each tumor were counted. BrdU incorporation is reported as percentage positive divided by total cell number. (D) Left: tumors were sectioned and stained for H&E, Slug, and AR expression. Right: total protein was isolated from tumors, and expression of AR and Slug is shown from 3 representative samples of each tumor type. Original magnification, ×20. Statistical analyses are representative of mean ± SEM. *P < 0.05; ***P < 0.001.

Figure 7. Slug enhances prometastatic phenotypes of PCa cells in vivo.

(A) Fertilized special pathogen–free eggs were incubated for 10 days at 38°C in a rotary humidified incubator. After 10 days of incubation, small holes were drilled over the air sac and near the allantoic vein. 2 × 10⁶ cultured human prostate adenocarcinoma cells (stable LNCaP-vec and LNCaP-Slug cell lines) were implanted onto the membrane in each egg. After sealing the windows, the eggs were incubated in a stationary incubator for 7 days and the embryos were sacrificed after 17 total days of incubation. The embryonic livers and lungs were harvested and analyzed for the presence of tumor cells using quantitative human Alu-specific PCR. (B) LN-SLUG or control cells (150,000 cells/mouse) were injected via the tail vein, after which whole organs were harvested, sectioned completely through; total number of fluorescent cells are reported/organ (left) and representative images shown (right). (C) C42-SLUG-GFP or C42-vec-RFP cells were injected into the left ventricle of nude mice along with fluorescent beads (used as a marker of proper injection). One hour after injection, organs were harvested, whole tissues were sectioned through, and total number of cells counted under a fluorescent microscope. Cells homing to the lung and liver are shown (right and quantified left). Scale bars: 200 μm. Error bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 8. The cyclin D1b–Slug network is conserved in advanced cancers.

(A) A panel of 108 CRPC cores were stained for cyclin D1b and Slug, and distribution of cyclin D1b and Slug staining as a function of intensity is plotted as a percentage of all samples (n = 118). Pathological determination of low-to-no staining was calculated as harboring an intensity score of 0–40, intermediate staining a score of 41–120, and high staining as greater than 120. (B) Left: representative staining of low (top) and high (bottom) cyclin D1b and Slug from matched samples of CRPC. Original magnification, ×5 (left); ×20 (right). (B) Right: correlation analyses plotted with Slug expression as a function of cyclin D1b intensity score was conducted using a 2-tailed Spearman’s correlation, and a line of best fit was generated using linear regression software. (C) Slug intensity scores were divided into the bottom and top 50% and matched Ki67 (percentage positive obtained from University of Tampere) scores are reported. Statistical analysis utilized Students 2-tailed t test to calculate differences between the 2 groups. *P < 0.05.