SUPPLEMENTARY INFORMATION
METHODS:
Cell culture and transfections
The MDA-MB-436 human breast cancer cell line was obtained from the ATCC and was grown in
accordance with their guidelines. For inhibitor studies, cells were treated for 24 hours in complete
media with 50μM SU5416 (Selleckchem), 40μM Perifosine (Sigma) or 1μM SCH772984
(Selleckchem), to inhibit activity of VEGFR2, AKT or Erk, respectively.
Transient knockdown of GPNMB in MDA-MB-436 cells was achieved by 48-hour
transfection (Lipofectamine 3000, Invitrogen) using indicated concentrations of an
ONTARGETplus
SMARTpool
of
4
GPNMB-targeted
siRNAs
(Dharmacon).
An
ONTARGETplus pool of 4 scrambled siRNAs was used as a transfection control. The transfection
was performed according to the manufacturer’s instructions.
RNA extraction, cDNA synthesis and quantitative Real-Time PCR
Triplicate RNA samples were extracted from BT549, 66cl4 and NIC cell lines at ~50% confluence
using RNeasy Mini Kits (Qiagen) and quantified using a spectrophotometer (Nanodrop ND-1000).
Total RNA (1μg per sample) was used to generate cDNA using a High Capacity cDNA Reverse
Transcription Kit (Applied Biosystems). Oligonucleotide primers were designed using Invitrogen
OligoPerfect software and pre-designed primers were identified in Primer Bank (Harvard
University) or RT-PCR Design (Roche Diagnostics). All primers were diluted to a concentration
of 100μM (Primer sequences can be found in Supplementary Table 2). RT-qPCR reactions were
performed on diluted cDNA (1:20) using Power SYBR Green Master Mix (Applied Biosystems)
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and 7500 Real Time PCR System (Applied Biosystems). Reactions were performed in triplicate
and GAPDH or β-actin primers were used as a control for mouse and human genes, respectively.
Data is represented as the mean of the fold change of the three independent sets of cDNA
calculated according to the following formula:
Efficiency of target primers + 1 (average value of target primers – average of corresponding target wells)
Efficiency of control primers + 1 (average value of control primers – average of corresponding control wells)
Microarray analysis
GPNMB-expressing clones, along with empty vector controls, were established in human BT549
and mouse 66cl4 breast cancer cell lines. Total RNA from two independent GPNMB-expressing
clones, along with two empty vector clones, was isolated from both the BT549 and 66cl4 model
systems. Total RNA was amplified, labeled and hybridized to whole genome 44K Agilent gene
expression arrays as previously described.1 Data were normalized based on the Lowess
normalization included in the Genespring software (Agilent technologies). A parametric test was
used to compare the GPNMB expressing clones and the parental cell lines (p<0.05). Genes that
were differentially expressed between GPNMB-expressing cells and VC cells, which exhibited a
fold change greater than 2, were chosen for further analysis. Both the 66cl4 and BT549 breast
cancer cell models were compared to identify genes that are commonly regulated by
overexpression of mouse and human GPNMB.
Immunohistochemistry analysis
Tissue samples were fixed, processed and stained with Ki67, cleaved caspase-3 or CD31
antibodies as previously described.2
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Adhesion blocking assays
To determine if GPNMB-mediated adhesion to fibronectin was specifically mediated through the
α5β1 receptor, 1x105 BT549 cells were trypsinized and incubated on ice for 1 hour with indicated
concentrations of blocking antibody targeting α5β1, α2β1 (Millipore) or control isotype antibody
(BD BioSciences) diluted in 1x PBS. Subsequently, cells were seeded onto 24-well plates precoated with fibronectin (BD Biosciences) and allowed to adhere for 1 hour at 37ºC. The plates
were processed and analyzed as previously described.3
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REFERENCES:
1. Rose, A.A., et al., Osteoactivin promotes breast cancer metastasis to bone. Mol Cancer
Res, 2007. 5(10): p. 1001-14.
2. Northey, J.J., et al., Distinct phosphotyrosine-dependent functions of the ShcA adaptor
protein are required for transforming growth factor beta (TGFbeta)-induced breast cancer
cell migration, invasion, and metastasis. J Biol Chem, 2013. 288(7): p. 5210-22.
3. Tabaries, S., et al., Claudin-2 is selectively enriched in and promotes the formation of
breast cancer liver metastases through engagement of integrin complexes. Oncogene,
2011. 30(11): p. 1318-28.
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FIGURE LEGENDS
Supplementary Table 1: Identification of commonly regulated genes in response to GPNMB
expression in the 66cl4 and BT549 breast cancer cell models. The list of 17 genes, with the
corresponding fold change in GPNMB-expressing versus VC cells, which were similarly regulated
in both the 66cl4 and BT549 cell models is shown. Neuropilin-1 was selected for further validation
and analysis.
Supplementary Table 2: A list of the shRNA sequences against mouse and human NRP-1 and
primer sequences used for mutagenesis and RT-qPCR.
Supplementary Table 3: A list of all the antibodies used in the current study.
Supplementary Figure 1. Gene expression changes induced by GPNMB in murine and breast
cancer cells. (a) Immunoblot analysis showing GPNMB expression in clonal (Cl) cell lines
generated in independent mouse (66cl4) and human (BT549) breast cancer models. An
immunoblot for α-Tubulin is included as a loading control. (b) Whole genome 44K Agilent gene
expression array analysis comparing two GPNMB expressing clonal cell lines and two VC cell
lines for both 66cl4 and BT549 cell models. Differentially expressed genes in GPNMB-expressing
versus VC cells were filtered on a fold change of 2 or greater and a P value of < 0.05. The
intersection between differentially expressed genes in the 66cl4 and BT549 cell systems revealed
17 genes that were commonly regulated by GPNMB.
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Supplementary Figure 2. GPNMB overexpression in BT549 and 66cl4 breast cancer cells results
in numerous transcriptional changes. Commonly regulated genes identified by microarray analysis
were validated by RT-qPCR analysis. Transcript levels of CLU (Clusterin) (b), Serpine2 (c), ANK1
(d), AHR (e) and PNRC1 (f) were increased in GPNMB-expressing BT549 cells compared to cells
containing an empty vector. Average transcript levels in GPNMB-expressing cells represent the
combined result of 4 independent BT549 clones displaying elevated GPNMB expression. CLU
(Clusterin) (h) and Serpine2 (i) were also found to be increased at the mRNA level in response to
GPNMB overexpression in 66cl4 cells, as determined by taking the average transcript
measurement of two independent GPNMB-expressing 66cl4 clones. RT-qPCR analyses displaying
elevated GPNMB mRNA levels in BT549 (a) and 66cl4 (g) cells are shown. (*, p < 0.05; **, p <
0.01; ***, p < 0.001). (n = 3)
Supplementary Figure 3. Neuropilin-1 expression is decreased in response to GPNMB
knockdown in MDA-MB-436 breast cancer cells. GPNMB expression was transiently diminished
in the MDA-MB-436 cell line using 2 different siRNA concentrations. Control cells were
transfected with a pool of scrambled non-targeting siRNAs. After 48 hours, cells were lysed,
processed and probed for NRP-1 and GPNMB. Α-Tubulin was used as a loading control.
Supplementary Figure 4. Neuropilin-1 expression levels in NIC cells following shRNAmediated knockdown. Immunoblot analysis of NRP-1 expression in NIC VC and GPNMB-WT
cells reveals a substantial knockdown of NRP-1 with the targeting shRNA. GPNMB levels remain
unchanged following NRP-1 knockdown. Α-Tubulin serves as a loading control.
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Supplementary Figure 5. Immunohistochemistry analysis of proliferation, apoptosis and
angiogenesis markers in tumors derived from NIC cell lines. Primary tissue was harvested from
NIC VC/NRP-1High, VC/NRP-1Low, GPNMB-WT/NRP-1High and GPNMB-WT/NRP-1Low tumors
and proliferation (a), apoptosis (b) and endothelial cell recruitment (c) was assessed by
immunohistochemistry. Results represent the averages of 10 images/sample taken from 5
independent primary tumor tissue samples. (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
Supplementary Figure 6. Immunohistochemistry for the macrophage marker F4/80. Primary
mammary tumor tissue from NIC VC/NRP-1High, VC/NRP-1Low, GPNMB-WT/NRP-1High and
GPNMB-WT/NRP-1Low was harvested, sectioned and stained for F4/80 to assess macrophage
infiltration. Quantification of F4/80 staining is presented in Figure 2b. Images representative of
the tumor margin (low magnification) and tumor core (inset) areas are shown. Scale bar represent
50μm and applies to all low magnification images.
Supplementary Figure 7. NRP-1 expression following stable knockdown in BT549 human breast
cancer cells. BT549 vector control (VC) and GPNMB-WT-expressing cells harboring a scrambled
shRNA (shLMP) or two independent shRNAs targeting the 3’ UTR region of NRP-1 (shRNA#1
and shRNA#2) were probed for NRP-1 and GPNMB. GPNMB levels remain unchanged in BT549
cells harboring an NRP-1 knockdown. Α-Tubulin serves as a loading control.
Supplementary Figure 8. GPNMB potentiates VEGF signaling specifically through the VEGFR2
axis. To inhibit VEGFR2 activity, BT549 GPNMB-WT cells were treated with 50μM Semaxanib
or DMSO vehicle. After 24 hours of inhibitor or control treatment, cells were stimulated with
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25ng/mL VEGF for the indicated times and activation of downstream signaling pathways was
assessed by immunoblot analyses for pVEGFR2, VEGFR2, pERK, ERK, pAKT, AKT, NRP-1
and V5-tagged GPNMB. Α-Tubulin serves as a loading control. Band intensity was quantified and
the ratios of phosphorylated to total protein were calculated and normalized to the first lane
(DMSO, no VEGF).
Supplementary Figure 9. GPNMB increases NRP-1 levels via AKT-mediated, but not ERKmediated, signaling. BT549 cells expressing VC or GPNMB were treated with 40μM Perifosine
(a) or 1μM SCH772984 (b) for 24 hours to inhibit AKT or ERK, respectively. (a) Immunoblot
analyses of NRP-1, pAKT, AKT and GPNMB (V5-tagged) in BT549 cells treated with Perifosine
(AKT inhibitor). (b) Immunoblot analyses of NRP-1, pFRA-1, FRA-1 (an ERK substrate) and
GPNMB (V5-tagged) in BT549 cells treated with SCH772984 (ERK inhibitor). Α-Tubulin was
used as a loading control.
Supplementary Figure 10. The GPNMB-mediated increase in NRP-1 expression requires its
RGD domain. Immunoblot analyses showing NRP-1 and GPNMB levels in BT549 (a) and 66cl4
(b) cells expressing VC, GPNMB-WT, GPNMB-ΔCYT and GPNMB-RGDmut constructs. ΑTubulin serves as a loading control.
Supplementary Figure 11. Fibronectin adhesion of GPNMB-expressing BT549 cells is
specifically mediated through the α5β1 integrin receptor. BT549 VC and GPNMB-WT cells were
incubated with control isotype antibodies or increasing concentrations of antibodies recognizing
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α2β1 or α5β1 integrin receptors. Area occupied by BT549 cells was quantified by taking the pixel
count from 5 independent images/well.
Supplementary Figure 12. Expression levels of GPNMB mutants in NIC breast cancer cells.
GPNMB levels in NIC VC, GPNMB-WT, GPNMB-ΔCYT and GPNMB-RGDmut cell lines were
assayed by immunoblot. Α-Tubulin serves as a loading control.
Supplementary Figure 13. Immunohistochemical analysis of NIC tumors expressing GPNMB
mutant constructs. (a) Proliferation of NIC VC, GPNMB-WT, GPNMB-ΔCYT and GPNMBRGDmut tumors was assessed by Ki67 staining. (***, p < 0.001) (b) Cleaved Caspase-3 was used
as a marker to determine the percentage of apoptosis in NIC tumors. (*, p < 0.05; **, p < 0.01;
***, p < 0.001) (c) Endothelial cell recruitment was determined by performing CD31 staining.
(***, p < 0.001). The average quantification of 10 representative images taken from each tumor
sample is shown (n = 5).
Supplementary Figure 14. Immunohistochemistry for the macrophage marker F4/80.
Macrophage infiltration was examined in NIC VC, GPNMB-WT, GPNMB-ΔCYT and GPNMBRGDmut expressing mammary tumors by performing immunohistochemistry for F4/80.
Representative images of the tumor margin (low magnification) and tumor core (inset) areas are
shown. Scale bar represent 50μm and applies to all low magnification images.
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