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Home , BCCIP, CDC42BPA, DIS3L, Forkhead box C1, GRK6, IKBKAP

Supporting Information

Gupta et al. 10.1073/pnas.1525387114 SI Methods objective, and a Canon EOS 40D digital single-lens reflex cam- Chemical Approach for Dissection of the Breast TIC Program. Only era. Correlation analysis for G3BP2 and SART3 staining in the selected compounds that target MDA-MB-231 LM2 cells in adjacent sections then was performed to assess colocalization. combination with paclitaxel (0.2 μM for 48 h) were used. Of Image pairs were subjected to automated contrast enhancement 60,000 compounds screened, 256 were selected for the second and then were registered using Photoshop (Adobe). Any non- screening step. To eliminate toxic compounds, nonmalignant overlapping image areas created in the registration process were cells were treated with these small molecules. One hundred eliminated by cropping both images. Next, the images were seventeen nontoxic compounds were selected for further analy- subjected to correlation analysis using ImageJ (https://imagej. sis. Cell viability (MTT) assays of modified MDA-MB-231 can- nih.gov/). Using the LAB color space, the images were seg- cer cells with five different concentrations (0.12, 0.37, 1.11, 3.33, mented to create binary images of the stained regions (thresh- and 10 mol/L) were carried out. To determine which bind olds for pass were L: 0–154; A: 0–255; BN: 123–255). An to this compound, TurboBeads carboxy nanoparticles (Turbo- automated procedure then quantified the number of positive Beads) were conjugated to compound C108 for 20 min per the pixels in 100 × 100 pixel (23 × 23 μm) regions of interest placed ’ manufacturer s protocol, followed by overnight immunoprecipi- in a grid pattern over the images. The corresponding datasets for tation at 4 °C. from metastatic cancer cells were pulled the two images then were subjected to statistical analysis to de- down with nanoparticles conjugated to compound C108 and with termine the correlation coefficient and P value using Prism nanoparticles alone as a control. Purified proteins were sepa- (GraphPad). rated on an 8% agarose gel, and a few bands that bound to compound C108 but not to the control were cut out from the gel RNA-Binding Immunoprecipitation. A RNA-binding protein and analyzed by mass spectrometry. Mass spectrometry analy- immunoprecipitation kit (Millipore) was used per the manufac- sis revealed 44 proteins that bound to compound C108. We turer’s protocol. BT-474 cells were treated with 0.3 μM paclitaxel obtained shRNA-expressing lentivirus for each pull-down pro- for 1 h. RIP buffer was used to collect protein, followed by im- tein and infected modified MDA-MB-231 cells. The MTT assays munoprecipitation overnight at 4 °C with G3BP2 - were performed with shRNA stable cell lines treated with non- lethal doses of paclitaxel. Only shRNA G3BP2 made cells sen- A/G magnetic beads. A magnetic separator was used to separate sitive to treatment with paclitaxel. To confirm that compound the beads from the buffer, and the beads were washed three – C108 binds to G3BP2, immunoprecipitation with magnetic times with RIP wash buffer. The RNA protein complex was nanoparticles bound to compound C108 and nanoparticles alone digested with proteinase K for 30 min at 55 °C, and RNA was (control) was carried out. purified with a phenol:chloroform:isoamyl alcohol extraction. mRNA was purified with the RNAeasy Kit (Qiagen) and was Histologic Analysis of Clinical Samples. Adjacent tissue sections analyzed with the Affymetrix microarray. were stained for G3BP2 and SART3, respectively, and RGB Only mRNA data that showed an in increase of 1.6 or higher images were acquired using an Olympus BX40 microscope, 10× after treatment compared with the controls were selected.

Gupta et al. www.pnas.org/cgi/content/short/1525387114 1of11 Fig. S1. Identification of anti-TIC chemical compounds. (A) Schematic illustration of screening approaches to identify anti-TIC chemical compounds. Of the 60,000 compounds screened, we selected 117 for the second screening step. We studied their efficacy at five different concentrations (0.12, 0.3, 1.11, 3.33, and 10 M). We then selected 78 of these 117 compounds that showed dose-dependent repression of cancer and that were nontoxic to normal human umbilical vein endothelial cells. (B) The molecular structures of the selected compounds.

Gupta et al. www.pnas.org/cgi/content/short/1525387114 2of11 Fig. S2. G3BP2 rescues the decrease of ALDEFLUOR mediated by compound C108. (A) Magnetic beads were combined with compound C108 followed by immunoprecipitation of proteins to compound C108. Interactive proteins were analyzed by mass spectroscopy. The construction of stable cell lines with repressed interacting proteins was followed by the detection of paclitaxel-sensitive cells. (B) MDA-MB-453 cells show a G3BP2-mediated rescue of the decrease in ALDEFLUOR phenotype induced by C108+paclitaxel. Cells were treated with vehicle or with 1 μM compound C108 plus 0.1 μM paclitaxel or with 1 μM compound C108 plus 0.1 μM paclitaxel plus endogenous G3BP2 and were analyzed by flow cytometry.

Gupta et al. www.pnas.org/cgi/content/short/1525387114 3of11 Fig. S3. Correlations of G3BP2 expression and outcomes of patients. (A–C) The Kaplan–Meier Plotter was used to analyze the correlation of G3BP2 with outcomes of breast cancer patients. Division by the median showed worse recurrence-free survival (A), distant metastasis-free survival (B), and overall survival (C). (D–H) Increased G3BP2 levels correlate with worse recurrence-free survival in different subtypes of breast cancer: basal type (D), + ER-negative (E), ER-positive (F), luminal A (G), and luminal B (H). (I) In patients with HER2 breast cancer, increased G3BP2 levels show improved survival.

Gupta et al. www.pnas.org/cgi/content/short/1525387114 4of11 Fig. S4. Colocalization of G3BP2 and PABPC1. (A) Stable knockdowns of G3BP2 were created with shRNA in the MDA-MB-453, BT-474, and MDA-MB-231 cell lines. (B) MDA-MB-231 cells were treated with 1 μM of paclitaxel for 30–60 min and were stained with G3BP2 (red) and PABPC1 (green) antibodies. (C) BT-474 cells and BT-474 cells with G3BP2 down-regulation were treated with 1 μM of paclitaxel for 30–60 min, stained with G3BP2 (red) and PABPC1 (green) anti- bodies, and imaged by multispectral confocal imaging.

Gupta et al. www.pnas.org/cgi/content/short/1525387114 5of11 A

scr-shRNA shRNA G3BP2 scr-shRNA

50 cells 500 cells shRNA G3BP2

B scr-shRNA shRNA G3BP2 cells 4 10 cells 5 10

Fig. S5. G3BP2 knockdown has a pronounced effect on tumor cell self-renewal. (A) Significantly higher numbers of cells BT-474 cells with silenced G3BP2 expression were required to form tumors. (B) ELDA for the tumor-forming frequency of G3BP2 shRNA MDA-MB-453 cells and control scr-shRNA MDA-MB-453 cells in NOD-SCID mice.

Fig. S6. Down-regulation of G3BP2 SG protein in human breast cancer cells leads to a decrease of SART3. (A) Fluorescent immunocytochemical staining was performed to determine the expression of SART3 in G3BP2-depleted and control cells. (Magnification: 200×) (Scale bars, 20 μm.) (B) Immunocytochemistry was performed to determine the expression of SART3 in G3BP2-depleted and control cells. (Magnification, 200×.) (Scale bar, 20 μm.)

Gupta et al. www.pnas.org/cgi/content/short/1525387114 6of11 A

MDA-MB-453 B

scr-shRNA shRNA SART3 ) (µm spheres Number of S Sphere size BT-474 ) (µm spheres Number of Sphere size

Fig. S7. G3BP2 regulates Oct-4, Nanog, and SART3 expression. (A) Western blot analysis was performed to detect OCT-4 and Nanog expression in SART3- knockdown breast cancer cell lines. (B) Representative images and analysis of mammosphere formation observed in SART3-silenced (white bars) and control (black bars) BT-474 and MDA-MB-453 cells. Data represent mean ± SD; *P < 0.05; **P < 0.005. (Magnification: 200×.)

Fig. S8. No change in or E-cadherin levels is seen after repression of G3BP2 in BT-474 cells. The effect of G3BP2 silencing on EMT markers was assessed using Western blot analysis. No change in vimentin or E-cadherin was noted. TWIST1 and Slug protein levels were decreased after knockdown of G3BP2.

Gupta et al. www.pnas.org/cgi/content/short/1525387114 7of11 Table S1. Proteins that bind to anticancer stem-cell compound C108 Protein coverage determined N Protein Protein name Matches by count, %

1 SNW1 SNW domain-containing protein 1 17 201/536 37.5 2 USP39 U4/U6-snRNP-associated protein 2 9 127/565 22.5 3 CSNK2A1 Casein 2, alpha 1 polypeptide 9 65/397 16.4 4 IGF2BP1 Insulin-like growth factor 2 mRNA-binding protein 1 9 104/577 18.0 5 PPP2R1B Isoform 1 of /threonine-protein 2A 5 72/601 12.0 65 kDa regulatory subunit A beta isoforms. 6 PTPN9 Protein tyrosine phosphatase, nonreceptor type 9 6 73/593 12.8 7 CRKL Crk-like protein 12 120/303 39.6 8 TBRG4 TBRG4 cDNA FLJ56153 6 65/642 10.1 9 GNL3 Isoform 2 of Guanine nucleotide-binding protein-like 3 10 124/537 23.1 10 Serine/threonine- PLK1 6 65/603 10.8 11 SHOC2 -rich repeat protein SHOC-2 8 91/582 15.6 12 IGF2BP2 Insulin-like growth factor 2 mRNA-binding protein 2 6 73/599 12.2 13 CDKN2AIP CDKN2A interacting protein 8 108/580 18.6 14 METAP2 Methionine aminopeptidase 2 13 123/478 25.7 15 LYRIC MTDH Protein LYRIC 9 128/582 22.0 16 ARCN1 Coatomer subunit delta variant 2 8 84/552 15.2 17 GRK6 Isoform GRK6A of G protein-coupled kinase 6 9 109/576 18.9 18 PABPC1 Isoform1 of Polyadenylate-binding protein1 15 161/636 25.3 19 IKIP Isoform 1 of Inhibitor of nuclear factor kappa-B kinase-interacting protein 7 72/350 20.6 20 HDGF Hepatoma-derived growth factor 5 25/240 11.2 21 OSGEP Probable O-sialoglycoprotein endopeptidase 6 55/335 14.6 22 MAP2K6 Isoform 1 of Dual specificity -activated protein kinase kinase 6 8 68/334 20.4 23 NACC1 Nucleus accumbens-associated protein 1 11 136/548 24.8 24 MAPK1 Mitogen-activated protein kinase 1 9 87/369 24.2 25 RGS19 Regulator of G protein signaling 19 9 100/354 28.2 26 RANGAP1 Ran GTPase-activating protein 1 9 122/587 18.0 27 G3BP2 Ras GTPase-activating protein-binding protein 2 11 101/482 21.0 28 G3BP1 Ras GTPase-activating protein-binding protein 1 22 246/466 52.8 29 PPP1Ca 1, catalytic subunit, alpha isozyme 4 34/309 11.0 30 MAP3K7IP1 Mitogen-activated protein kinase kinase kinase 7-interacting protein 1 5 64/507 12.7 31 PIP5K1A Phosphatidylinositol-4-phosphate 5-kinase, type I, alpha 6 62/500 12.4 32 PPP2R1A , regulatory subunit A, alpha 6 70/509 13.8 33 API5 Isoform 2 of inhibitor 8 102/504 20.2 34 MPP7 MAGU.K. p55 subfamily member 7 kinase 11 144/576 25.0 35 ORC2L Origin recognition complex subunit 2 10 125/577 21.7 36 RIC8A Resistance to inhibitors of 8 homolog A 6 55/537 10.2 37 RBBP5 cDNA FLJ59722, highly similar to Retinoblastoma-binding protein 5 4 64/573 11.2 38 MAPKAPK3 MAP kinase activated protein kinase 3 7 65/382 17.0 39 CDK9 Isoform1 of cell division protein kinase 9 9 122/372 32.8 40 ARMCX3 Armadillo repeat-containing X-linked protein 3 10 89/379 23.5 41 THOC6 Isoform 3 of THO complex subunit 6 homolog 18 76/296 25.7 42 PIH1D1 PIH1 domain-containing protein 1 6 77/290 26.6 43 UFD1L Isoform short of fusion degradation protein 1 homolog 5 35/307 11.3 44 PSTPIP2 Isoform 1 of -serine-threonine phosphatase-interacting protein 2 5 66/334 19.8

Gupta et al. www.pnas.org/cgi/content/short/1525387114 8of11 Table S2. mRNAs identified as highly expressed (1.6-fold increase in expression) compared with control Fold change Gene Gene name

3.69 DIS3L DIS3 mitotic control homolog (S. cerevisiae)-like 3.62 MAP3K1 Mitogen-activated protein kinase kinase kinase 1, E3 ubiquitin protein 3.45 MYCBP2 binding protein 2, E3 ubiquitin protein ligase 3.22 DSTYK Dual serine/threonine and tyrosine protein kinase 3.22 INPP5F Inositol polyphosphate-5-phosphatase F 3.11 NOTCH2NL, NBPF10 Notch 2 N-terminal like, neuroblastoma breakpoint family member 21-like, neuroblastoma breakpoint family, member 10 2.96 NRK Nik related kinase 2.92 PTPN13 Protein tyrosine phosphatase, nonreceptor type 13 (APO-1/CD95 (Fas)-associated phosphatase) 2.89 JMY Junction mediating and regulatory protein, 2.84 HDGFRP2 Hepatoma-derived growth factor-related protein 2 2.77 PGR 2.76 SACS Spastic ataxia of Charlevoix-Saguenay (sacsin) 2.73 PLCB4 C, beta 4 2.67 MAP3K5 Mitogen-activated protein kinase kinase kinase 5 2.64 PHF10 PHD finger protein 10 2.63 NFIB /B 2.62 GREB1 Growth regulation by estrogen in breast cancer 1 2.57 MAP2K4 Mitogen-activated protein kinase kinase 4 2.54 PRKDC Protein kinase, DNA-activated, catalytic polypeptide 2.5 CDK5RAP2 CDK5 regulatory subunit associated protein 2 2.43 BRCC3 BRCA1/BRCA2-containing complex, subunit 3 2.41 IKBKAP Inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase complex-associated protein 2.38 NFKB1 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 2.37 ATR Ataxia telangiectasia and Rad3 related 2.36 CDC42BPB CDC42 binding protein kinase beta (DMPK-like) 2.35 N4BP2 NEDD4 binding protein 2 2.34 APAF1 Apoptotic peptidase activating factor 1 2.33 ETAA1 Ewing tumor-associated antigen 1 2.31 PPIP5K1 Diphosphoinositol pentakisphosphate kinase 1 2.31 PRKAR2A Protein kinase, cAMP-dependent, regulatory, type II, alpha 2.3 ATM, NPAT Ataxia telangiectasia mutated, nuclear protein, ataxia-telangiectasia 2.29 MINA MYC induced nuclear antigen 2.28 PIKFYVE Phosphoinositide kinase, FYVE finger containing 2.28 PPIP5K1 Diphosphoinositol pentakisphosphate kinase 1 2.26 PHKB kinase, beta 2.26 PPP2R3A Protein phosphatase 2, regulatory subunit B’’, alpha 2.24 INPP4A Inositol polyphosphate-4-phosphatase, type I, 107kDa 2.24 MAP4K3 Mitogen-activated protein kinase kinase kinase kinase 3 2.24 MAPK7 Mitogen-activated protein kinase 7 2.23 SYK Spleen tyrosine kinase 2.18 PPIP5K2 Diphosphoinositol pentakisphosphate kinase 2 2.17 PPIP5K1 Diphosphoinositol pentakisphosphate kinase 1 2.15 NIN Ninein (GSK3B interacting protein) 2.14 PTPN14 Protein tyrosine phosphatase, nonreceptor type 14 2.13 BLM Bloom syndrome, RecQ helicase-like 2.11 DAPK3 Death-associated protein kinase 3 2.11 GRB14 Growth factor receptor-bound protein 14 2.11 PIK3CA Phosphoinositide-3-kinase, catalytic, alpha polypeptide 2.10 PIK3C2B Phosphoinositide-3-kinase, class 2, beta polypeptide 2.08 AKAP13 A kinase (PRKA) anchor protein 13 2.08 PPTC7 PTC7 protein phosphatase homolog (S. cerevisiae) 2.07 MADD MAP-kinase activating death domain 2.07 MAP2K6 Mitogen-activated protein kinase kinase 6 2.07 TIAM1 T-cell lymphoma invasion and metastasis 1 2.07 TP53BP1 Tumor protein p53 binding protein 1 2.07 TP63 Tumor protein p63 2.06 CDK14 Cyclin-dependent kinase 14 2.06 JAK1 Janus kinase 1 2.06 SART1 Squamous cell carcinoma antigen recognized by T cells 2.05 BBS2 Bardet-Biedl syndrome 2

Gupta et al. www.pnas.org/cgi/content/short/1525387114 9of11 Table S2. Cont. Fold change Gene Gene name

2.05 CDK5RAP1 CDK5 regulatory subunit associated protein 1 2.05 HDAC8 Histone deacetylase 8 2.04 CDK13 Cyclin-dependent kinase 13 2.04 IRS1 Insulin receptor substrate 1 2.03 CAMK2B Calcium/calmodulin-dependent protein kinase II beta 2.02 DICER1 Dicer 1, type III 2.01 CMIP c-Maf inducing protein 2.01 FANCI Fanconi anemia, complementation group I 2.01 GAS8 Growth arrest-specific 8 2.01 KDM5C Lysine (K)-specific demethylase 5C 2.01 PTPN3 Protein tyrosine phosphatase, nonreceptor type 3 2.01 RBL2 Retinoblastoma-like 2 (p130) 1.99 HPS3 Hermansky–Pudlak syndrome 3 1.99 KDM1A Lysine (K)-specific demethylase 1A 1.99 PI4K2A Phosphatidylinositol 4-kinase type 2 alpha 1.98 HPS6 Hermansky–Pudlak syndrome 6 1.97 CDC42BPA CDC42 binding protein kinase alpha (DMPK-like) 1.97 HELQ Helicase, POLQ-like 1.97 MAP3K4 Mitogen-activated protein kinase kinase kinase 4 1.97 MARK1 MAP/ affinity-regulating kinase 1 1.95 TP53BP2 Tumor protein p53 binding protein, 2 1.94 EPG5 Ectopic P-granules autophagy protein 5 homolog (C. elegans) 1.94 IBTK Inhibitor of Bruton agammaglobulinemia tyrosine kinase 1.94 NOTCH2NL Notch 2 N-terminal like 1.93 BCAR3 Breast cancer anti-estrogen resistance 3 1.93 KDM3A Lysine (K)-specific demethylase 3A 1.93 RIPK1 Receptor (TNFRSF)-interacting serine-threonine kinase 1 1.92 PPFIA1 Protein tyrosine phosphatase, receptor type, f polypeptide (PTPRF), interacting protein (liprin), alpha 1 1.92 PTPN21 Protein tyrosine phosphatase, nonreceptor type 21 1.91 EPC1 Enhancer of polycomb homolog 1 (Drosophila) 1.89 MDC1 Mediator of DNA-damage checkpoint 1 1.88 GADD45G Growth arrest and DNA-damage-inducible, gamma 1.88 IGFBP6 Insulin-like growth factor binding protein 6 1.88 NOTCH2NL Notch 2 N-terminal like 1.87 ATRX Alpha thalassemia/mental retardation syndrome X-linked 1.87 HIGD1B HIG1 hypoxia inducible domain family, member 1B 1.86 PRKAR1B Protein kinase, cAMP-dependent, regulatory, type I, beta 1.85 PHF2 PHD finger protein 2 1.85 PI4KA Phosphatidylinositol 4-kinase, catalytic, alpha 1.85 RBBP8 Retinoblastoma binding protein 8 1.84 DROSHA Drosha, ribonuclease type III 1.84 E2F8 factor 8 1.84 NEK9 NIMA (never in gene a)- related kinase 9 1.84 PFKFB2 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 1.84 TAOK3 TAO kinase 3 1.83 BAG1 BCL2-associated athanogene 1.83 CDC14B CDC14 cell division cycle 14 homolog B (S. cerevisiae) 1.82 GREB1L Growth regulation by estrogen in breast cancer-like 1.80 BRCA1 Breast cancer 1 1.80 PPP2R3C Protein phosphatase 2, regulatory subunit B’, gamma 1.80 PRKCD , delta 1.79 ADK Adenosine kinase 1.79 PPIP5K1 Diphosphoinositol pentakisphosphate kinase 1 1.79 PTPRM Protein tyrosine phosphatase, receptor type, M 1.79 SP100 SP100 nuclear antigen 1.78 HOXB1 B1 1.78 PPP2R3B Protein phosphatase 2, regulatory subunit B’’, beta 1.77 CCL3 Chemokine (C-C motif) ligand 3 1.77 PHF17 PHD finger protein 17

Gupta et al. www.pnas.org/cgi/content/short/1525387114 10 of 11 Table S2. Cont. Fold change Gene Gene name

1.77 PI4KAP2, PI4KAP1, PI4KA Phosphatidylinositol 4-kinase, catalytic, alpha pseudogene 2, phosphatidylinositol 4-kinase, catalytic, Alpha pseudogene 1, phosphatidylinositol 4-kinase, catalytic, alpha 1.77 PIP5K1B Phosphatidylinositol-4-phosphate 5-kinase, type I, beta 1.76 CDC45 Cell division cycle 45 homolog (S. cerevisiae) 1.76 FOXC1 Forkhead box C1 1.76 MAP4K4 Mitogen-activated protein kinase kinase kinase kinase 4 1.76 RBBP6 Retinoblastoma binding protein 6 1.76 RIOK1 RIO kinase 1 (yeast) 1.76 YY1 YY1 1.75 HIPK1 Homeodomain interacting protein kinase 1 1.75 MAPKAPK5 Mitogen-activated protein kinase-activated protein kinase 5 1.74 CCNB2 Cyclin B2 1.74 CCNE1 Cyclin E1 1.74 WRN Werner syndrome, RecQ helicase-like 1.72 CCL19 Chemokine (C-C motif) ligand 19 1.72 HRASLS HRAS-like suppressor 1.72 IRS2 Insulin receptor substrate 2 1.71 AKT3 v-Akt murine thymoma viral oncogene homolog 3 (, gamma) 1.71 BAP1 BRCA1 associated protein-1 (ubiquitin carboxyl-terminal ) 1.71 BCCIP BRCA2 and CDKN1A interacting protein 1.71 CDC14C CDC14 cell division cycle 14 homolog C (S. cerevisiae) 1.70 MAPK14 Mitogen-activated protein kinase 14 1.69 BRCA2 Breast cancer 2, early onset 1.69 PIK3R1 Phosphoinositide-3-kinase, regulatory subunit 1 (alpha) 1.68 CDKL5 Cyclin-dependent kinase-like 5 1.68 PABPC1L2B, PABPC1L2A Poly(A) binding protein, cytoplasmic 1-like 2B, poly(A) binding protein, cytoplasmic 1-like 2A 1.68 PLK4 Polo-like kinase 4 1.68 SMAD5 SMAD family member 5 1.67 MARK3 MAP/microtubule affinity-regulating kinase 3 1.66 MAP2K5 Mitogen-activated protein kinase kinase 5 1.65 NFATC3 Nuclear factor of activated T-cells, cytoplasmic, -dependent 3 1.65 NKIRAS2 NFKB inhibitor interacting Ras-like 2 1.65 PALB2 Partner and localizer of BRCA2 1.64 SART3 Squamous cell carcinoma antigen recognized by T cells 3 1.63 PPIP5K1 Diphosphoinositol pentakisphosphate kinase 1 1.62 CDC26 Cell division cycle 26 homolog (S. cerevisiae) 1.62 PPP3CA Protein phosphatase 3, catalytic subunit, alpha isozyme 1.61 NEK8 NIMA (never in mitosis gene a)- related kinase 8 1.61 PIK3R2 Phosphoinositide-3-kinase, regulatory subunit 2 (beta) 1.61 PPIP5K1 Diphosphoinositol pentakisphosphate kinase 1 1.61 PPM1M Protein phosphatase, Mg2+/Mn2+ dependent, 1M

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