PROX1-mediated epigenetic silencing of SIRT3 contributes to proliferation and glucose metabolism in colorectal cancer

Prospero-related homeobox 1 (PROX1) is a homeobox transcription factor known to promote malignant transformation and stemness in human colorectal cancer (CRC). However, the biological function of PROX1 in metabolic rearrangement in CRC remains unclear. Here, we aimed to uncover the relationship between the expression profile and role of PROX1 and CRC cell glucose metabolism and to elucidate the underlying molecular mechanism. PROX1 expression was significantly upregulated in human CRC tissues and positively associated with the maximum standardized uptake value (SUVmax), a measure of tissue 18-fluoro-2-deoxy-D-glucose uptake and an indicator of glycolysis and tumor cell activity, in patients with CRC. Knockdown of PROX1 suppressed CRC cell proliferation and glucose metabolism in vitro and in vivo. Mechanistically, through a physical interaction, PROX1 recruited EZH2 to the SIRT3 promoter and inhibited SIRT3 promoter activity. Moreover, PROX1 or EZH2 knockdown decreased cell glycolysis by targeting SIRT3. Clinically, high PROX1 expression combined with low SIRT3 expression predicted poor prognosis in patients with CRC. Thus, our study suggests that the PROX1-EZH2 complex positively regulates cell proliferation and glucose metabolism by engaging SIRT3 in CRC, which may serve as a promising therapeutic strategy for CRC.

agarose beads (Sigma, St. Louis, MO) preequilibrated in RIPA buffer. The beads were washed with RIPA buffer, and bound proteins were eluted using 3xFlag peptide (Sigma). Both eluants and postelution beads were boiled in loading buffer, resolved by SDS-PAGE and silver stained. Lysates from HEK293T cells transfected with empty vector were used as controls and processed in parallel. Bands specific to the pFlag-PROX1 transfection were excised and subjected to mass spectrometry analysis on ABI 4700 MALDI TOF/TOF.

Real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis
Total RNA was extracted with TRIzol (Invitrogen) according to the manufacturer's instructions. Real-time qRT-PCR and western blot assays were performed as previously described 19 .
GST-tagged and His-tagged proteins were further purified using GST or Ni-NTA Sefinose Resin kits (Sangon Biotech, China) following the manufacturer's protocol.
Briefly, a mixture of the protein extract with binding buffer was added to columns filled with resin, which has a high affinity for GST-tagged or His-tagged recombinant proteins. Nonspecifically bound proteins were eliminated by washing the resin until the absorbance of the flowthrough fraction at 280 nm approached the baseline value. Then, recombinant proteins were eluted from the resin with elution buffer. Protein elution was monitored by measuring the absorbance of the fractions at 280 nm. Finally, the eluted proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Immunoprecipitation and coimmunoprecipitation
Cells were collected and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime) containing a protease inhibitor cocktail (Roche Diagnostics).
Whole-cell lysates (2 mg) were precleared with 30 μL of protein G beads (Life Technologies), and 2 μg of isotype-matched IgG control or antibodies was then added and incubated for 2 hours on a rocking platform. Immunoprecipitants were collected by centrifugation and were then resolved by SDS-PAGE.

Cell proliferation and colony formation assay
The CCK-8 assay and colony formation assay were performed as previously described 19 .

Glycolysis analysis
The cellular glycolysis rate was determined by a Glucose Uptake Colorimetric Assay Kit and Lactate Colorimetric Assay Kit (BioVision). RT-PCR was used to determine the expression levels of glycolytic enzymes in the cells 20 .

Analysis of ATP production
The ENLITEN ATP Assay System (Promega; FF2000) was used according to the manufacturer's instructions. Cells were harvested by scraping and resuspended in PBS. The cell suspension was divided into unequal aliquots. Part of the cell suspension was mixed with 5% trichloroacetic acid (TCA). The remaining cells were used for the cell number calculation. Tris-acetate buffer (pH 7.75) was then added to neutralize the TCA and to dilute the TCA to a final concentration of 0.1%.
The diluted sample (40 mL) was added to an equal volume of rL/L reagent (Promega; FF2000). Then, luminescence was measured. The ATP standard (Promega; FF2000) was serially diluted to generate a regression curve for calculating ATP concentrations in individual samples. The relative ATP concentration was determined and normalized to that of the control cells, which was designated as 1.

Cellular mitochondrial function was measured using a Seahorse XF Cell Mito Stress
Test Kit and a Bioscience XF96 Extracellular Flux Analyzer according to the manufacturer's instructions. The glycolytic capacity was determined using a Glycolysis Stress Test Kit (ab222946, Abcam) according to the manufacturer's instructions. Briefly, 4 ×10 4 cells were seeded in 96-well plates and incubated overnight. After washing the cells with Seahorse buffer (DMEM with phenol red containing 25 mmol/L glucose, 2 mmol/L sodium pyruvate, and 2 mmol/L glutamine), 175 mL of Seahorse buffer plus 25 mL each of 1 mmol/L oligomycin, 1 mmol/L FCCP, and 1 mmol/L rotenone was automatically injected to measure the OCR. Then, 25 mL each of 10 mmol/L glucose, 1 mmol/L oligomycin, and 100 mmol/L 2-deoxyglucose was added to measure the ECAR. The OCR and ECAR values were calculated after normalization to the cell number and are plotted as the means ± SDs.

Animal studies
Animal experiments were approved by the Ethics Committee at Fudan University Shanghai Cancer Center (FUSCC) and performed as previously described 19,21 .
Sections of the specimens (4 μm) were cut for IHC staining.

Immunofluorescence staining
Immunofluorescence staining was performed as previously described 20 . E. Glucose uptake, lactate production, and ATP levels in EZH2-knockdown and control HCT116 and SW480 cells were determined as described in the Materials and Methods. * P < 0.05; according to Student's t test.

Supplementary
F. ECAR and OCR in EZH2-knockdown and control HCT116 and SW480 cells.
G. RT-qPCR analysis of the indicated rate-limiting enzymes in EZH2-knockdown and control HCT116 and SW480 cells. * P < 0.05; according to Student's t test.
H. Representative images (upper panel) and distribution analysis (lower panel) of EZH2 immunohistochemical staining in CRC and normal colorectal samples