Overexpression of SRC‐3 promotes esophageal squamous cell carcinoma aggressiveness by enhancing cell growth and invasiveness

Abstract Steroid receptor coactivator‐3 (SRC‐3), a transcriptional coactivator for nuclear receptors and other transcription factors, plays an important role in the genesis and progression of several cancers. However, studies investigated the role of SRC‐3 in esophageal squamous cell carcinomas (ESCCs) are limited, and the role of SRC‐3 in tumor progression remains unclear. We examined the expression of SRC‐3 in 8 ESCC cell lines and 302 human ESCC tissues by qPCR, Western blot, and immunohistochemistry. In addition, ESCC cell lines were subjected to proliferation and invasion assays, tumorigenicity assay, flow cytometry assay, qPCR, Western blot, and Chromatin Immunoprecipitation assay to investigate the role of SRC‐3 in cancer progression. SRC‐3 was overexpressed in 48% of cases and correlated with poor overall (P = 0.0076) and progression‐free (P = 0.0069) survival of surgically resected ESCC patient. Cox regression analysis revealed that SRC‐3 is an independent prognostic marker. Furthermore, we found that activation of insulin‐like growth factor (IGF)/AKT) was involved in the SRC‐3 on the cell growth and invasiveness in two ESCC cell lines, Eca109 and EC18 cells. SRC‐3 overexpression is clinically and functionally relevant to the progression of human ESCC, and might be a useful molecular target for ESCC prognosis and treatment.


Immunohistochemistry (IHC)
IHC staining was performed on 4-µm TMA sections rehydrated through gradient alcohols. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide for 15 min and antigen were retrieved or 10 min in 10mmol/L citrate buffer (pH 6.0) at 98°C. Nonspecific binding was blocked with 10% normal rabbit serum for 20 min.
The TMA slides were incubated with anti-SRC-3 (a monoclonal antibody directed at amino acids 376-389 of SRC-3, Transduction Laboratories, San Jose, CA, 1:50 dilution) for 60 min at 37°C in a moist chamber. Subsequently, the slides were sequentially incubated with biotinylated rabbit antimouse immunoglobulin at a concentration of 1:100 for 30 min at 37°C and then reacted with a streptavidin-peroxdase conjugate for 30 min at 37°C and 3'-3' diaminobenzidine as a chromogen substrate. The nucleus was counterstained using Meyer's hematoxylin.
The negative control was performed by replacing the primary antibody with a normal murine IgG. Known immunostaining positive slides were used as positive controls.
Positive expression of SRC-3 in ESCC and normal esophageal mucosa cells was primarily a nuclear pattern. The malignant and non-malignant tissues were scored for SRC-3 by assessing the site of positive staining in the nucleus. The staining of the nuclei of the normal esophageal mucosa ranged from 0-10% of epithelium with positive staining, thus, overexpression of SRC-3 was scored when more than 10 percent of tumor cells were positively stained in the nuclei, normal expression level of SRC-3 when less than 10 percent of tumor cells were positive.

Vectors, Retroviral Infection, and stable cell line selection
To silence endogenous SRC-3, the DNA oligonucleotides encoding short hairpin RNA (shRNA, such as 5'-ggatcc ACCCTGAGAGCTTTATTACttcaagaga GTAATAAAGCTCTCAGGGT tttttaagctt-3', with sequences in capitals targeting human SRC-3 mRNA), were synthesized, annealed, and subcloned into retroviral Eca109 and EC18 were infected with culture supernatants from individual 293FT cells at a multiplicity of infection of 10 in the presence of 2ug/mL polybrene (Sigma-Aldrich). Stable cell lines expressing SRC-3 shRNA were selected for 10 days with 0.25µg/mL puromycin 48 h after infection. Following the selection, cell lysates prepared from the pooled population of cells in sampling buffer were fractionated on SDS-PAGE for western blot detection of respective protein levels.

RNA extraction, cDNA synthesis, and qPCR
Total RNA was isolated using TRIzol reagent (Invitrogen) as the manufacturer instructed. Three micrograms of total RNA was used for a reverse transcription reaction with Moloney murine leukemia virus RT and oligo(dT)18 primers; cDNAs were diluted 10-fold, and a 5mL of dilution was used for the PCRs. Gene sequences were amplified in the presence of SYBR Green fluorophore and detected using ABI µg of PMSF/mL, protease inhibitor cocktail) for 30 min on ice with constant vigorous vortexing. The debris was cleared by centrifugation at 12,000 rpm for 10 min at 4°C.
Lysates were boiled in gel loading buffer and separated on 5% to 8% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gradient gels and transferred to nitrocellulose membranes. After blocked with 5% non-fat milk in PBS with 0.2% Tween-20, membranes were incubated overnight at 4°C with primary antibodies listed as below, followed by horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. All blots were developed with enhanced chemiluminescence Western blotting detection reagent (Amersham Biosciences). Signal intensities were determined by densitometry and normalized using anti-β-actin antibodies.

5-bromo-2′-deoxyuridine (BrdU) incorporation assays
For BrdU incorporation, cells were seeded on poly-L-lysine coated glass coverslips in six-well plates and BrdU was added to the culture medium 1h before fixation. The cells were fixed in 95% methanol at 4°C, denatured in 2 mol/L of HCl and stained with mouse anti-BrdU fluorescein conjugate (Roche, Basel, Switzerland), according to the manufacturer's recommendations. DAPI was used as the nuclear counterstain. Microscopic fields were selected randomly and ≥200 cells were counted under each condition. BrdU-positive cells were calculated as the percentage of total cells in each field.

Methyl thiazolyl tetrazolium (MTT) assays
Cell viability assays and growth curve analysis were evaluated by MTT assay as described elsewhere. Briefly, a total of 1x10 3 cells were seeded in 96-well plates and MTT (5 mg/mL) was added to each well every 24 h. The plates were incubated at 37°C for 4 h, and then 100µL dimethylsulfoxide (DMSO) were added to each well to lyse the cells. The absorbance at 490 nm was then recorded by a microplate reader (Bio-Tek).

Colony formation and Soft argar assays
Cells were collected with trypsin and seeded into 100-mm dishes at a concentration of 1 ×10 3 after counting. The plates were incubated at 37°C in a humidified incubator containing 5% CO 2 . When formation of colonies was visible (2-3 weeks), they were fixed with 4% formaldehyde, stained with crystal violet, and counted.
A soft agar colony formation assay was used to assess the anchorage-independent growth ability of cells. Cells were resuspended in 0.33% soft agar with 1 mL of growth medium (Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum) and layered onto 0.66% solidified agar in six well plates. The soft agar colonies were allowed to grow for two weeks at 37°C.
Colonies greater than 100 µm in diameter at low magnification (×100) were scored as positive and counted at four points on each well.

Flow cytometry assay
Flow cytometry assay was done by propidium iodide staining. Cells were grown to 70% to 80% confluence, then harvested and fixed overnight at 4°C in 70% ethanol.
After washed with PBS, cells were incubated with 5µg/µL propidium iodide and 50µg/µL RNase in PBS for 30 min at 37°C. Each sample was analyzed by fluorescence-activated cell sorter analysis (FACS) (BD, San Jose, CA, USA). The cell cycle distribution was established by plotting the intensity of the propidium iodide signal, which reflects the cellular DNA content.

Wound healing assay
Cell migration was assessed by measuring the movement of cells into a scraped, acellular area created by a 10µl pipette tip, and the spread of wound closure was observed after and photographed at 0, 10 and 20 h under a microscope.

Transwell assay
Invasion assays were performed in 24-well Bio-Coat Matrigel Invasion Chambers

Chromatin Immunoprecipitation (ChIP) assay
ChIP assay were performed using a Magna ChIP Assay Kit (Millipore, Billerica, MA) according to the manufacturer's protocol. In briefly, cells were treated with 1% formaldehyde for 10 min to cross-link associated protein to DNA, lysed, and then sonicated. The crude chromatin solution was diluted and incubated at 4°C with specific antibodies overnight, or negative control mouse immunoglobulin G. PCR was performed using 5 µL of purified ChIP DNA for 28 cycles with promoter-specific primers. Primer sequences are listed as below: