AF1q is a novel TCF7 co-factor which activates CD44 and promotes breast cancer metastasis.

AF1q is an MLL fusion partner that was identified from acute myeloid leukemia (AML) patients with t (1; 11) (q21; q23) chromosomal abnormality. The function of AF1q is not yet fully known, however, elevated AF1q expression is associated with poor clinical outcomes in various malignancies. Here, we show that AF1q specifically binds to T-cell-factor-7 (TCF7) in the Wnt signaling pathway and results in transcriptional activation of CD44 as well as multiple downstream targets of the TCF7/LEF1. In addition, enhanced AF1q expression promotes breast cancer cell proliferation, migration, mammosphere formation, and chemo-resistance. In xenograft models, enforced AF1q expression in breast cancer cells also promotes liver metastasis and lung colonization. In a cohort of 63 breast cancer patients, higher percentages of AF1q-positive cancer cells in primary sites were associated with significantly poorer overall survival (OS), disease-free survival (DFS), and brain metastasis-free survival (b-MFS). Using paired primary/metastatic samples from the same patients, we demonstrate that AF1q-positive breast cancer cells become dynamically dominant in the metastatic sites compared to the primary sites. Our findings indicate that breast cancer cells with a hyperactive AF1q/TCF7/CD44 regulatory axis in the primary sites may represent "metastatic founder cells" which have invasive properties.


Mammosphere assay
Cells were trypsinized to obtain a single cell suspension, and then diluted to a concentration of 1×10 4 cells/ml for HMLE or 2×10 4 cells/ml for other cells in Opti-MEM (Invitrogen). 1 ml of 1% (w/v) methylcellulose medium was added per well to 24-well ultra-low attachment surface plates (Costar). 200 µl of prepared cell suspension was seeded into each well containing 1% (w/v) methylcellulose medium and mixed vigorously. Media was replenished every 3-4 days for 10-21 days.

RNA and miRNA extraction and RT-qPCR analysis
The total RNA extracted with mirVana miRNA isolation kit (Invitrogen) was reverse transcribed using High Capacity cDNA Reverse Transcription kits (Applied Biosystems) according to the manufacturer's instructions. qPCR was performed on an ABI 7500 System (Applied Biosystems), using TaqMan Universal PCR Master Mix (Applied Biosystems). The following sense (S) and antisense (AS) primers were used to amplify CD44 by qPCR; S: 5'-TCCAACACCTCCCAGTATGACA-3', AS: 5'-GGCAGGTCTGTGACTGATGTACA-3'. miRNAs were detected by RT-qPCR using the TaqMan miRNA Assay. mRNA and miRNA29b expression was normalized to HPRT1 and RNU48, respectively. All samples were run in triplicate and mean value and s.d. were calculated.

Western blot and Cellular fraction
For blots of whole-cell lysates, cells were lysed directly in GLB buffer (2% SDS, 10% glycerol and 50 mM Tris, pH 6.8), boiled, and separated by electrophoresis on a 4 -12% SDS-PAGE gradient gel. Proteins were transferred to PVDF membrane (Millipore) and blocked in 5% skim milk (Bio-Rad) in 0.05% PBST. Rabbit monoclonal anti-AF1q antibody was co-developed with Epitomics. Anti-CD44, TCF7 and β-actin monoclonal antibodies were purchased from Cell Signaling Technology, Inc. After the appropriate antibody incubations, an enhanced chemiluminescence (Denville) system was used for developing blots.

Cellular fractionation was performed using the NE-PER™ Nuclear and Cytoplasmic
Extraction Kit (Thermo Scientific) according to the manufacturer's instructions. Protein concentration was evaluated in each fraction using a BCA kit (Thermo Scientific) after isolation and then performed Western blotting as described above using AF1q antibody. GAPDH and Histone H3 (Santa Cruz) were used for internal loading control.

Anoikis Assay
Cells were dissociated and plated (2×10 5 cells/well) into a 6-well Ultra-Low Attachment Cluster Dish (Costar) with normal growth media for 24 hours to induce anoikis. Apoptosis was analysed using Dead Cell Apoptosis Kit with Annexin V APC and SYTOX Green (Invitrogen). Apototic cells and dead cells were detected using annexin V expression and SYTOX nucleic acid staining by flow cytometry, respectively. All samples were analyzed triplicate and cells plated normal culture plate were used as a control.

Co-immunoprecipitation (Co-IP) assays
MCF10a cells were fractionated and nuclear fractions were incubated with anti-TCF7 antibody on a rocker at 4°C overnight. Immunocomplexes were pulled down by incubating with Protein A-Sepharose (Cell Signaling) for 1 h at 4°C, followed by washing 3 times with ice-cold lysis buffer to eliminate non-specific interactions. Protein A-sepharose-bound immunocomplexes were then resuspended in SDS-PAGE loading buffer, boiled for 10 min, and analyzed by Western blot as described above.

Wnt pathway activity assay
Dual-Luciferase reporter assays were performed using a TOP/FOP reporter system (Milipore).

CD44 promoter luciferase assay
To construct reporter plasmids, 890 bases of a CD44 (-908/-118) promoter fragment were cloned to between XhoI and HindIII sites of the pGL4-Luciferase reporter plasmid (Promega) (5). Luciferase activity was measured as described above.
Reaction mixtures containing no nuclear extract were also incubated with labeled probes as negative control samples. Each sample was loaded onto a 6% polyacrylamide gel in 0.5X Tris/Borate/EDTA (TBE) buffer. For the antibody super-shift analysis, 1 μg of antibody was added to the nuclear extracts prior to incubation with the Alexa-488 labeled probe for 30 min. In competition analysis, 100-fold molar excess of the unlabeled double-strand oligonucleotide was added to the reaction mixture prior to the addition of the labeled probe. Samples were electrophoresed until the dye had reached 1 inch from the bottom of the gel and then scanned with the Typhoon 9410 imager (GE Healthcare).

RNA-seq
We used Cuffdiff 24 to perform differential expression testing to detect the differential expressed genes. Genes with q<0.05 were defined to be statistically differentially expressed. Mice were imaged weekly using the IVIS imaging system for 4 weeks. Mice were euthanized and autopsied after 4 weeks post-cell injection. Mice were fed 200 mg/kg of doxycycline daily.

Immunohistochemistry
Tissue samples were fixed in 4% paraformaldehyde, embedded in paraffin, and then 4-µm sections were prepared. Sections were de-waxed and a steamer pre-treatment in Tris/EDTA buffer (DAKO) was performed. Endogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide in PBS. For blocking steps, avidin (Sigma-Aldrich), biotin (Sigma-Aldrich) in PBS, and a super block (IDlabs Biotechnology) were used. Rabbit monoclonal AF1q antibody in a 1:200 dilution was incubated at 4°C overnight. IHC detection was performed with the IDetect Super Stain System HRP (IDlabs Biotechnology). Specific signals were amplified using 3-amino-9-ethylcarbazole (IDlabs Biotechnology) under visual control, followed by counterstaining with hematoxylin.