RUNX2 regulates breast cancer cell metabolism
Supplemental Figure Legends
Supplemental Figure S1: Breast cancer cell models expressing RUNX2. (A) Inducible RUNX2
expression in ER+ MCF7 cells was established using the BD™-Tet-Off System. RUNX2 and ER
expression were measured with specific antibodies. RUNX2+ MCF7 cells expressed lower levels of ER
(mean = 0.56 ± 0.04) relative to RUNX2- cells (mean = 0.74 ± 0.01; p < 0.018; t-test). (B) Triple negative
Hs578t cells were infected with lentiviral shRNA targeting endogenous RUNX2. After stable selection,
clones with reduced RUNX2 expression relative to parental cells were analyzed (Hs578t/53.5; Hs578t/55.5)
while another clone (Hs578t/54.5) served as a control knockdown clone that did not show a decrease in
RUNX2 expression. MCF7.TetOFF (RUNX2–) or cells expressing RUNX2 (RUNX2+) (C) or Hs578t
parental or shRNA knockdown cells (D) were grown for 3 days in complete media. Cell growth was
measured using crystal violet staining (Absorbance 540nm). Each point is the mean ± SD of triplicate
samples; p > 0.05).
Supplemental Figure S2: SIRT6 expression in Hs578t knockdown clones. (A) Comparison of SIRT6
expression in parental (Hs578t), control knockdown 54.5, or Runx2 knockdown (55.5) Hs578t cells. Cells
were culture in full media containing 10%FBS and 25mM glucose (FM) or in glucose starvation media
containing 2%FBS for 8hr (S). Nuclear extracts were examined for RUNX2 and SIRT6 expression by
Western blot. Relative SIRT6 protein expression normalized to actin (NIH Image-J) from scanned blots is
indicated under each lane. (B) Overexpression of SIRT6 in breast cancer cells. Hs578t cells (RUNX2+)
were transfected with empty vector (Vector) or cDNA expression vector encoding human SIRT6. After a
brief selection (1 week), nuclear extracts were isolated and analyzed for SIRT6 and RUNX2 expression by
Western blotting. SIRT6 relative density in arbitrary units (AU) normalized to actin was calculated from
three determinations using NIH Image-J; * indicates p < 0.05 relative to Vector. (C) Glucose starvationresistant cells (Hs578t.LG) from Hs578t cells were obtained in low glucose (0.5mM with 5% FBS) for 4 days.
Surviving cells were expanded in standard cell culture media (DMEM+10% FBS) for 10-14 days. Cells were
analyzed for RUNX2 and GLUT1 expression. Significant differences in RUNX2 (p < 0.01; t-test) and GLUT1
(p < 0.06; t-test) expression for LG clones compared to parental cells were calculated by Image-J. (D)
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RUNX2 regulates breast cancer cell metabolism
Hs578t parental and LG2 cells were treated with different concentrations of glucose as indicated and
analyzed for SIRT6 expression after 16hr. Relative SIRT6 protein expression normalized to actin (NIH
Image-J) from scanned blots is indicated.
Supplemental Figure S3: Mitochondrial OCR in MCF7 and Hs578t cells . (A) MCF7 RUNX2 + or
RUNX2 – cells were compared for OCR using the Seahorse metabolic flux analyzer. FCCP was used to
depolarize the inner mitochondrial membrane and inhibitors of mitochondrial ETC were used as in Figure 5.
Treatments include: FCCP + pyruvate (Pyr) ( 0.4μM + 10mM), FCCP (0.1μM), FCCP (0.1μM), and
antimycin-A (1μM). Cell protein was extracted after determination of OCR and was similar for RUNX2+
(10.68 ± 0.17 μg) and RUNX2– (10.10 ± 2.07 μg) from n = 11 wells per sample (p > 0.05)
(B) Hs578tP (Parental) or control knockdown (Hs578t/54.5) cells were compared for OCR using the
Seahorse metabolic flux analyzer. As in Figure 5, FCCP (0.75μM), pyruvate (10mM), and antimycin-A
(1μM) were used to treat cells. Oligomycin (2.5nM) inhibition indicates that in these cells >95% of the
oxygen consumption was linked to mitochondrial ATP synthesis. Inset shows RUNX2 expression in
Hs578t/54.5 (control knockdown) and Hs578t/55.5 (RUNX2 knockdown) cells compared to parental cells.
(C) Knockdown of SIRT6 in breast cancer cells. Hs578t/55.5 (low RUNX2 expression) cells were
transfected with scrambled (Control) siRNA or three different SIRT6-specific siRNA oligonucleotides (siRNA
A, siRNA B, and siRNA C) from Origene. Levels of SIRT6 were determined by Western blot with specific
SIRT6 antibody. Relative density in arbitrary units (AU) represents the mean from three determinations
normalized to actin (NIH Image-J); * indicates p < 0.05 relative to Control. (D) Hs578t/55.5 cells were
transfected with control or siRNA C targeting SIRT6. SIRT6 and RUNX2 protein levels are shown and
compared relative to actin. The Western blot was overexposed to visualize the low levels of RUNX2 in 55.5
knockdown cells. Band density relative to control is indicated.
Supplemental Figure S4: Hif1 and SIRT6 expression in response to RUNX2. (A) MCF7 cells cultured
in the presence (+Dox; RUNX2-) or absence of doxycycline (-Dox; RUNX2+) were starved in the absence of
glucose for 16hr to lower Hif1 levels and then treated with 5mM glucose for the indicated times. Some
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RUNX2 regulates breast cancer cell metabolism
cells were treated with the SIRT inhibitor, sirtinol (Sirt; 10μM) and 5mM glucose for 8hr. Cell lysates were
prepared and the expression of Hif1α, SIRT6, or RUNX2 was measured by Western blotting. Gels were
stripped and reprobed for ß-actin (loading control). Relative density of scanned Western blot (NIH Image-J)
was normalized to actin and expressed as arbitrary units (AU). (B) MCF7 cells cultured in +Dox or -Dox to
induce RUNX2 were cultured in 5mM glucose, placed in a hypoxia chamber, and exposed to 1%O2 for the
indicated times. Cells were lysed and the expression of Hif1α was measured by Western blotting. Relative
band intensities were determined with NIH Image-J software from these Western blots. Essentially similar
results were obtained from two additional determinations
Supplemental Figure S5: Ubiquitination of SIRT6 and transcriptional activity of RUNX2 in HEK293T
cells. (A) Cells were transfected with SIRT6 and/or RUNX2 expression vectors for 24hr and treated with
MG132 for 4hr. Nuclear extracts were isolated and immunoprecipitated with SIRT6-specific antibody
followed by Western blotting using anti-Ubiquitin or SIRT6 antibodies. (B) HEK293 cells were transfected
with the indicated plasmids (Flag empty vector, Flag.RUNX2, Flag.SIRT6, or SIRT6.prom.Luc) for 24hr prior
to incubation in low glucose (5mM) for 6hr. Relative GLuc:SEAP luciferase units were measured from
conditioned media. Differences in SIRT6 promoter activity after RUNX2 transfection compared to Flag
vector are indicated (NS; ANOVA). Differences in activity from RUNX2 + SIRT6 expression relative to
SIRT6 + Flag are indicated (*p < 0.05; ANOVA).
Supplemental Figure S6: Clinical correlates, RUNX2, and metabolic profiling in BC patients with IDC.
Tissues from patients admitted to the University of Maryland Greenebaum Cancer Center with invasive
ductal carcinoma were analyzed for GLUT1 and RUNX2 expression by immunohistochemistry (IHC). Scale
bar = 400μm for all panels. Inset magnification shows areas with cytoplasmic, membrane, nuclear, or
negative staining. Tumor profiling for patients with IDC was performed by the Department of Anatomic
Pathology (University of Maryland School of Medicine) and included ER/PR status, proliferative index (Ki67),
and standard uptake values (SUV) from 18F-DG positron emission tomography (FDG-PET) scans.
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RUNX2 regulates breast cancer cell metabolism
Supplemental Figure S7: Expression of RUNX2, GLUT1, and ER in human BC. (A) Panels show IHC
representative photos of normal or stage III tissue stained with H & E or specific antibodies recognizing
RUNX2 or Glut1. Controls used secondary antibody alone (2º Ab) and negative tissue stain for GLUT1 or
RUNX2. Scale bar = 100μm. (B) Results from TMA of normal, stage I (fibroadenoma/ductal carcinoma in
situ – FA/DCIS), stage II, stage II/III, and stage III patients. Panels show IHC representative photos of tissue
expressing RUNX2 or GLUT1 vs stage: cytoplasmic GLUT1 (GLUT1C), membrane-localized GLUT1
(GLUT1M), cytoplasmic RUNX2 (RUNX2C), and nuclear RUNX2 (RUNX2N). The Spearman correlation
coefficient was used to determine whether total RUNX2 expression was different across the five groups
(normal, FA/DCIS, stage II, stage II/III, and stage III). Significant differences between RUNX2 and GLUT1
across these groups were noted (p < 0.0001). A negative correlation between RUNX2 and cytosolic GLUT1
in normal tissues (r = –0.75) and a possible positive correlation between RUNX2 and membrane-bound
GLUT1 in stage III cancers (r = 0.42) were also noted. Scale bar = 100μm. Inset magnification shows
cytoplasmic, membrane, or nuclear localization. (C) Association between total RUNX2 and ERα. Using the
Spearman correlation coefficient, RUNX2 total and ER were assessed for each group: Normal (n = 16);
FA/DCIS (n = 7); Stage II (n = 88); Stage II/III (n = 14); Stage III (n = 16). A negative correlation between
RUNX2 and ER in normal tissue and in stage III tumors was noted (r = –0.48).
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