CDKL2 promotes epithelial-mesenchymal transition and breast cancer progression.

The epithelial-mesenchymal transition (EMT) confers mesenchymal properties on epithelial cells and has been closely associated with the acquisition of aggressive traits by epithelial cancer cells. To identify novel regulators of EMT, we carried out cDNA screens that covered 500 human kinases. Subsequent characterization of candidate kinases led us to uncover cyclin-dependent kinase-like 2 (CDKL2) as a novel potent promoter for EMT and breast cancer progression. CDKL2-expressing human mammary gland epithelial cells displayed enhanced mesenchymal traits and stem cell-like phenotypes, which was acquired through activating a ZEB1/E-cadherin/β-catenin positive feedback loop and regulating CD44 mRNA alternative splicing to promote conversion of CD24(high) cells to CD44(high) cells. Furthermore, CDKL2 enhanced primary tumor formation and metastasis in a breast cancer xenograft model. Notably, CDKL2 is expressed significantly higher in mesenchymal human breast cancer cell lines than in epithelial lines, and its over-expression/amplification in human breast cancers is associated with shorter disease-free survival. Taken together, our study uncovered a major role for CDKL2 in promoting EMT and breast cancer progression.


Immunofluorescence and immunohistochemistry assay
Cells grown on Lab-Tek II Chamber Slide (Thermo Scientific Nunc) were fixed with 4% paraformaldehyde/ PBS for 10 min, and permeabilized with 0.2% Trition-100/ PBS for 10 min before blocking with blocking buffer (PBS + 5% BSA + 0.1% Tween-20) for 1 h. Cells were then incubated with primary antibody (diluted 1:200) overnight at 4°C. Primary antibodies used were as follows: anti-E-cadherin (BD Bioscience Pharmingen), anti-βcatenin (Santa Cruz Biotechnology), anti-N-cadherin (BD Bioscience Pharmingen). After washing with PBS, secondary antibodies coupled to Alexa-488 or -594 (Invitrogen) were used in the dark at room temperature for 1 h. Cell nuclei were then visualized with DRAQ5 (Invitrogen). Slides were mounted with SlowFade Gold anti-fade reagent (Invitrogen). Images were capturedwith the Leica TCS SP5 Confocal Microscope and LAS AF software.
For tissue staining, slides with 5-μm sections of formalin-fixed, paraffin-embedded samples were deparaffinized and rehydrated before being subjected to antigen retrieval with 10 mM sodium citrate pH 6.0 for 20 min. Slides were then incubated with Peroxidase Suppressor (Thermo Scientific Pierce), Avidin/Biotin blocking solution (Vector Labs) and non-specific binding blocking buffer (PBS + 5% BSA + 0.1% Tween-20), followed by the corresponding primary antibody (overnight at 4°C) and HRPconjugated secondary antibody (1 h at room temperature). The immunohistochemistry reaction was developed with a DAB substrate Kit (Vector Labs) before slides were counterstained with hematoxilin and mounted in Di-N-butyl Phthalate in Xylenesolution (DPX, Sigma-Aldrich).

Reverse transcription and quantitative PCR analysis
RNA was isolated from cells using TRIzol reagent (Invitrogen). cDNA was generated by reverse transcription with iScript cDNA Synthesis Kit (Bio-Rad). Real time PCR was performed using SsoFast EvaGreen Supermix (Bio-Rad) in Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad) and PCR products were run on 2% agarose gels. For all RT-PCR analysis, either GAPDH or β-actin mRNA was used to normalize RNA input and expression levels were calculated according to the comparative C T method (ΔΔC T ). Primers sequence used to amplify genes were as follows:

Migration assay
Cells were starved by culturing in medium without growth factors overnight. After starvation, cells were split and 5 × 10 4 cells were seeded onto Transwell membrane inserts (24-well transwell plate containing 8 µm pore-size polycarbonate filters, Corning Costar) in growth factorsfree medium. Full media was added to the lower chamber. After 24 h incubation at 37°C, non-migrating cells were wiped from the upper side of the membrane. Cells that migrated to the lower surface of the membrane were fixed and stained with 0.5% crystal violet solution (in 95% ethanol). Duplicate inserts were used for each individual experiment, and five random microscopic fields were counted per insert.

Multilineage differentiation
For osteoblast differentiation, 1 × 10 4 cells were cultured in NH OsteoDiff Medium (MiltenyiBiotec) in 12-well plates for 35 days; and for adipocyte differentiation, 1 × 10 5 cells were cultured in NH AdipoDiff Medium (MiltenyiBiotec) for 42 days. The medium was replaced every 3 days. After culturing, committed osteogenic cells and adipogenic cells were detected as previously described [19,20]. Briefly, to test osteoblastic differentiation, cells were incubated with FAST BCIP/NBT (Sigma-Aldrich) to determine alkaline phosphatase (AP) activity, or analyzed by alizarin red S (Sigma-Aldrich) staining and silver nitrate (Von-Kossa) staining to determine calcium deposition and mineral deposition. As to adipocyte differentiation, oil red O dye (Sigma-Aldrich) staining was used to detect oil droplets formation.

Characterization of cell growth
Cells (300/well) were plated in 96-well plate. After 24 h, cells were gently washed with PBS and cultured with DMEM/F12 (1:1) medium supplemented with decreasing concentrations of growth factors for 12-14 days. Medium with full amounts of growth factors was regarded as 100% growth factors.The medium was replaced every 3 days. Cell proliferation was then measured using the AlamarBlue assay. Briefly, AlamarBlue reagent (invitrogen) was added to each well, and the plate was incubated at 37°C for 6 h. The plates were then read on the Tecan Infinite M1000 plate reader (TECAN Groups Ltd., Mannedorf, Switzerland) using an excitation wavelength of 535nm and an emission wavelength of 595nm. For drug resistance assay, all compounds were purchased from Sigma-Aldrich and dissolved in DMSO. Cells (3000/ well) were plated in 100 µl per well in 96-well plates. One day (24 h) after seeding, compounds were added in quadruplicate per concentration for each cell line. Cell viability was measured after 72 h using the AlamarBlue assay.

Promoter reporter luciferaseassay
Cells were transiently transfected with firefly luciferase reporter vectors using TransIT-LT1 Transfection Reagent (MirusBio). As internal control for transfection efficiency, renilla luciferase construct was cotransfected with reporter vectors. After 40 h, luciferase activity was assessed with Dual-Luciferase Reporter Assay System (Promega). All data were at least triplicate and analyzed using Student's t-test.

Animal experiments
NOD/SCID mice were purchased from Charles River Laboratories. All mouse procedures were approved by the Animal Care and Use Committees of University of Texas Health Science Center at Houston and performed in accordance with institutional polices.
HMLER-EV and HMLER-CDKL2 cells were transduced to express luciferase and in vitro luciferase assay was performed to ensure similar luciferase expression in both cell lines. The indicated number of cells were suspended in 30 µl 1:1 Matrigel (BD Bioscience):DMEM/F12 solution and then injected into the 4th inguinal mammary glands on both sides of NOD/ SCID mice. Mice were sacrificed after 12 weeks or when tumors reached a diameter > 1 cm.
Tumor size was measured with a slide caliper and the volumes were estimated according to the following formula: Tumor volume (mm 3 ) = π/6×L×W 2 , where L is length and W is width. For bioluminescence imagining, mice were anaesthetized using ketamin/xylazine and ZEB1-R  5′-GCTTCATCTGCCTGAGCTTC-3′   ZEB2-F  5′-ACAAGCCAGGGACAGATCA-3′   ZEB2-R  5′-GCCACACTCTGTGCATTTGA-3′ injected intraperitoneally with 150 mg/kg luciferin (Caliper Life sciences), and then primary tumor and metastasis were imaged using an IVIS Lumina II platform (Caliper Life sciences). Metastasis to lung, liver and chest bones were examined ex vivo upon sacrifice. Following culture in osteoblastic differentiation media, these cells were tested with FAST BCIP/NBT kit to determine alkaline phosphatase (AP) activity, or analyzed by alizarin red S staining and silver nitrate (Von-Kossa) staining to determine calcium deposition and mineral deposition.