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AB C Breast cancer Gas6 RNA Seq D E F Figure S1. AXL and GAS6 expression correlates with a mesenchymal signature. (A) Scatter plot representing the RNA.

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Presentation on theme: "AB C Breast cancer Gas6 RNA Seq D E F Figure S1. AXL and GAS6 expression correlates with a mesenchymal signature. (A) Scatter plot representing the RNA."— Presentation transcript:

1 AB C Breast cancer Gas6 RNA Seq D E F Figure S1. AXL and GAS6 expression correlates with a mesenchymal signature. (A) Scatter plot representing the RNA expression of GAS6 in 643 human cancer cell lines by RNA seq. The red line represents the mean expression within each tissue type. (B) Scatter plot demonstrating the correlation between AXL high and AXL low cells with Vimentin (VIM) and E-Cadherin (CDH1) expression. AXL high is defined as greater than the median (AXL Median RPKM = 4.73) expression across 643 cell lines and AXL low is less than the median. (C) Scatter plot demonstrating a significant difference in Vimentin expression in AXL high expressing cell lines compared to AXL low expressing cell lines as assessed using negative binomial distribution test (p= <0.0001). (D) Dot plot showing GAS6 expression in 46 breast cancer cell lines based on subtype. Highest expression of GAS6 is observed in triple negative breast cancer (TNBC). The black line represents the median expression within each tissue type. (E) Table illustrating AXL RPKM values within TNBC cell lines based on subtype. BL1, Basal-like 1; BL2, Basal-like 2; IM, Immunomodulatory; M, Mesenchymal; MSL, Mesenchymal stem-like; UNC, Unclassified. (F) Scatter plot demonstrating AXL expression in 147 lung cancer cell lines. AXL high and low expression was determined as greater or less than the median AXL expression in lung (AXL Median RPKM = 6.48).

2 Figure S2. AXL staining in TNBC tissues samples. IHC analysis of 26 TNBC tissues for AXL, E- Cadherin, and Vimentin. Insert shows higher magnification at 40x. (A) Patient number HP-31657, punctate cytoplasmic staining of AXL in tumor cells; focal vascular staining. (B) Patient number HP , membranous staining of AXL in a small subpopulation of tumor cells; stromal staining. (C) Patient number HP-31670, AXL: membranous staining of a small population of tumor cells; staining in stromal cells. (D) Detection of AXL, E-Cadherin, and Vimentin for all 26 TNBC samples.

3 Figure S3. TGF-β-induced EMT is reversible, and TKI resistance upon EMT is not due to drug efflux. (A) Immunoblot demonstrating in the HCC1954 cells that the epithelial marker E-Cadherin expression is restored and the mesenchymal marker snail expression decreases and AXL expression is modestly decreased, 8 days post-withdrawal of rh-TGF-β (red asterisk indicates rh-TGF-β withdrawal). (B) Immunoblot demonstrating a comparable suppression of pRTK and downstream signaling in the parental and mesenchymal rh-TGF-β-treated cell lines upon TKI treatment (Erlotinib 50nM 4h, Lapatinib 1μM 2h) in the PC9 (left panel) and HCC1954 (right panel) cells. AB

4 AB D Figure S4. AXL inhibitor R428 suppresses GAS6-induced pAXL, downstream signaling and invasion capacity. (A) Immunoblot demonstrating that R428 (1μM) suppresses GAS6-induced pAXL (GAS6 400ng/ml for 5 minutes). (B) Immunoblot demonstrating that R428 (1μM) suppresses basal and GAS6-induced pAKT and pS6 (GAS6 400ng/ml for 15 minutes) in MDA-MB-231 and HCC1143 (left panel) cells and no effect was observed with the AXL-negative BT-20 (right panel) cells. (C) Immunoblot demonstrating that R428 (1μM, 4h) decreases pAKT in both parental and mesenchymal PC9 cells. (D) Bar graph demonstrating that R428 (1μM) decreases invasion capacity in a 22-hour invasion assay in AXL-positive cell lines, HeLa, MDA-MB-231 and HCC1143; no effect on invasion capacity was observed with the AXL-negative cell line BT-20. Error bars represent the mean ± SEM. C

5 Figure S5: Inhibition of AXL does not re-sensitize erlotinib-resistant cells. (A) Immunoblot demonstrating that a decrease in E-Cadherin and an increase in vimentin expression in the Erlotinib-resistant (ERL-R) cell lines. Erlotinib treatment (1μM) for 6h does not alter E-Cadherin or vimentin expression. (B) Immunoblot demonstrating a decrease in AXL expression following siRNA knockdown (20nM) for 96h with four different AXL siRNA oligos. NTC siRNA serves as a control. (C) Bar graphs illustrating the effect of erlotinib (1μM) on cell viability in combination with AXL-Fc (10μg/ml) or YW327.6S2 (10μg/ml) for 72h in parental HCC4006 (left panel) and erlotinib-resistant HCC4006 (right panel) cell lines. (D) Bar graphs illustrating the effect of erlotinib (1μM) on cell viability in combination with AXL-Fc (10μg/ml) or YW327.6S2 (10μg/ml) for 72h in HCC827 erlotinib-resistant cells (ERL-R). (E) Immunoblot demonstrating a decrease in AXL expression following treatment with doxycycline in HCC827 ERL-R cells for 72h. (F) Cell viability assay demonstrating the effect of erlotinib (72h) in the presence (Dox+) and absence (Dox-) doxycycline in HCC827 ERL-R cells. Cells were also treated with or without doxycycline for 72h prior to erlotinib treatment. Error bars represent the mean ± SEM. (G) Bar graphs illustrating the effect of erlotinib (1μM) in combination with AXL-Fc (10μg/ml) or YW327.6S2 (10μg/ml) in HCC827 ERL-R cells for 72h on cell viability, in the absence (Dox-) (left panel) or the presence (Dox+) (right panel) of doxycycline. B C D EFG A

6 AXL β-actin HCC827-Axl HCC827 AB Figure S6: AXL over-expression or inhibition does not alter erlotinib sensitivity in the parental HCC827 cell line. (A) Immunoblot demonstrating an increase in AXL expression following overexpression in parental HCC827 cells. (B) Cell viability assay demonstrating the effect of erlotinib in AXL over-expressing cells for 72h. Error bars represent the mean ± SEM. (C) Bar graph illustrating the effect of erlotinib (1μM) in combination with AXL-Fc (10μg/ml) or the anti-AXL antibody YW327.6S2 (10μg/ml) for 72h on cell viability, in the AXL over-expressing cells. (D) Crystal violet staining demonstrating erlotinib-resistant HCC827 clones, following exposure to erlotinib in combination with AXL-Fc (10μg/ml) or YW327.6S2 (10μg/ml) for 41 days. Media, AXL-Fc, and YW327.6S2 were replenished every 3 or 4 days over a 41 day period. C D

7 Figure S7: Summary of Bliss scores and activity of MP-470, Erlotinib and Docetaxel (DTX) or combination treatment. (A) The delta Bliss was calculated based on the equation (A + B) – AB, where A (R428) and B (Drug X) are the fraction of growth inhibition at a fixed dose. Delta Bliss values are summed across the dose matrix to generate the Bliss sum. Values are from one representative experiment out of three independent experiments. (B) Cell viability assay demonstrating the effect of MP-470 (1μM) in combination with Docetaxel (DTX) in the HCC827 AXL-negative cell line cell line. Error bars represent mean ± SEM. (C) Cell viability assay demonstrating the effect of MP-470 (1μM) in combination with Docetaxel (DTX) in the HeLa cell line. Error bars represent mean ± SEM. A BC

8 A Paclitaxel (μM) Inhibition Bliss R428 (μM) B D Figure S8. R428 synergistically interacts with anti-mitotic agents and not with Doxorubicin or Cisplatin to reduce cell viability. (A) Cell viability assay demonstrating the effect of R428 (1μM) in combination with Paclitaxel in the HeLa cell line. Error bars represent mean ± SEM. (B) Drug matrix heat-map grid illustrating the percentage inhibition and delta Bliss for HeLa cells upon exposure to Paclitaxel in combination with R428. (C) Immunoblot demonstrating cleaved PARP following treatment with R428 (1μM) in combination with Paclitaxel (10nM) in HeLa cell line for 72h. (D) Bar graph representing Caspase-3/-7 activation following R428 (1μM) in combination with Paclitaxel (50nM) in HeLa cells. Error bars represent mean ± SEM. (E) Bar graph representing Caspase 3/7 activation following treatment with R428 (1μM) in combination with PHA (PHA, 150nM) in HeLa cells. Error bars represent mean ± SEM. (F) Drug matrix heat-map grid illustrating the percentage inhibition and delta Bliss for HeLa cells upon exposure to Doxorubicin in combination with R428. (G) Cell viability assay demonstrating the effect of R428 (1μM) in combination with Cisplatin in HeLa cells. Error bars represent mean ± SEM. FG C E

9 Figure S9. AXL inhibition in combination with anti-mitotic agents promotes mitotic death. (A) Table represents the fate of 100 individual cells following exposure to AXL-FC (3μg/ml), Docetaxel (DTX), and combination in a 72h microscopy assay. (B) Table represents the fate of 100 individual cells following exposure to MP-470 (1μM), Docetaxel (DTX), and combination in a 72h microscopy assay. (C) Table represents the fate of 100 individual cells following exposure to R428 (1μM), Gemcitabine (Gem), and combination in a 72h microscopy assay. (D) Scatter plot demonstrating the duration of completed mitosis (h) of 100 individual cells upon each drug treatment; R428 (1µM), Docetaxel (DTX), and combination in the MDA-MB-231 in a 72h assay. Red line indicates Mean. (E) Scatter plot demonstrating the duration of mitotic death interval (h) of 100 individual cells upon each drug treatment; R428 (1µM), Docetaxel (DTX) and combination in MDA-MB-231 in a 72h assay. Red line indicates Mean. (F) Scatter plot demonstrating a significant difference in the time of mitotic death (h) for 100 individual cells upon each drug treatment: R428 (1µM), Docetaxel (DTX; 10nM) and combination in the MDA-MB-231 (bottom panel) in a 72h assay as assessed using Student’s t-test (two-tailed, MDA-MB-231 p= ). Red line indicates Mean. (G) Table represents the fate of 100 individual cells following exposure to R428 (1µM), Docetaxel (DTX), and combination in a 72h microscopy assay. A B C D E F G

10 Figure S10. Suppression of pAKT, pS6 and pCDC2 following AXL inhibition. (A) Immunoblot illustrating enhanced suppression of pAKT and pS6 upon co-treatment with R428 (1μM) and Docetaxel (DTX, 3nM) in HeLa cells. (B) Immunoblot illustrating dephosphorylation of CDC2 by R428 treatment alone and in combination with docetaxel in MDA-MB-231 cells. (C) Immunoblot illustrating dephosphorylation of CDC2 by R428 in individual MDA-MB-231 xenograft tumors. pCDC2 (Tyr15) CDC2 Total DTX R428 ACB

11 Figure S11: AXL knock-down increases p21 expression. Immunoblot showing AXL, phospho-CDC2, total CDC2, p21, p53, phospho-CDC25C, total CDC25C, phospho WEE1, and total WEE1 levels following AXL siRNA knock-down for 72 hours and docetaxel (3nM) for a further 4h in HeLa cells.

12 Table S1. Mesenchymal PC9 cells are cross-resistant to a number of anti- cancer agents. Table represents the IC 50 values of parental PC9 and Mesenchymal PC9 (rh-TGF-β treated) cells.

13 Supplemental Reagents and Procedures Reagents The following antibodies were obtained from Cell signaling; phospho-ERK (#9101), total ERK (#9102), phospho-HER2 (#2247), total HER2 (#2242) and phospho-Tyrosine (#9411). Total EGFR was obtained from Santa Cruz Biotechnology (#SC-03) and phospho-EGFR was obtained from Abcam (#ab5644). The source of the anti-cancer agents is listed in Table S1. Immunoprecipitation of Axl Cell lysates were collected lysis buffer (10mM Tris pH 7.5, 150mM NaCl, 0.1% TX100), and 10% glycerol and supplemented with HALT protease and phosphatase inhibitor cocktail (Thermo Scientific). Lysate was incubate with Axl antibody (#4566 Cell signaling) conjugated agarose beads suspension (Pierce kit #44894). Immunodetection of phospho-Tyrosine was performed using standard protocols. Immunohistochemistry Immunohistochemistry (IHC) for Axl and Vimentin was performed on a Ventana Discovery XT autostainer and on a Ventana Benchmark XT for E-Cadherin (Ventana Medical Systems, Tucson, AZ). Formalin-fixed, paraffin-embedded whole tissue sections were deparaffinized and pretreated with CC1 solution (Ventana Medical Systems) followed by incubation with either Axl (1:2000, Clone 7E10, LifeSpan Biosciences, Seattle, WA), E-Cadherin (Clone 36, Ventana Medical Systems), or Vimentin (Clone V9, Ventana Medical Systems) mouse monoclonal antibody or naive mouse IgG (Clone MOPC, BD Pharmingen, San Jose, CA) for 16 (E-Cadherin and Vimentin) or 60 (Axl) minutes at 37ºC. Detection was performed with 16 minute OmniMap anti-mouse HRP incubation or Ultraview Detection System and DAB (Ventana Medical System) followed by counterstaining with Hematoxylin II (Ventana Medical System). Cell line authentication/quality control: Short Tandem Repeat (STR) Profiling STR profiles were determined for each line using the Promega PowerPlex 16 System. This was performed once and compared to external STR profiles of cell lines (when available) to establish cell line ancestry. Loci analyzed: Detection of sixteen loci (fifteen STR loci and Amelogenin for gender identification), including D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, AMEL, vWA, D8S1179 and TPOX. SNP fingerprinting: SNP genotypes are performed each time new stocks are expanded for cryopreservation. Cell line identity is verified by high-throughput SNP genotyping using Fluidigm multiplexed assays. SNPs were selected based on minor allele frequency and presence on commercial genotyping platforms. SNP profiles are compared to SNP calls from available internal and external data (when available) to determine or confirm ancestry. In cases where data is unavailable or cell line ancestry is questionable, DNA or cell lines are re-purchased to perform profiling to confirm cell line ancestry. SNPs analyzed: rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs12537, rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs , rs Mycoplasma Testing: All stocks were tested for mycoplasma prior to and after cells were cryopreserved. Two methods were used to avoid false positive/negative results: Lonza Mycoalert kit and Stratagene Mycosensor. Cell growth rates and morphology were also monitored for any batch-to-batch changes. Generation of stable cells lines: HCC827-AXL over-expressing cell line: 293GP cells were transfected with PQCXIP-AXL and pVSVG by lipofectamine 2000 for 48h. HCC827 parental cells were incubated with the harvested supernatant for 48h. AXL overexpressing cells were selected with puromycin (1μg/ml). HCC827 ERL-R-shRNA: 293GP cells were transfected with pHUSH-GW-AXL shRNA and pVSVG by lipofectamine HCC4006 ERL-R cells were incubated with the harvested supernatant for 48h and cells were selected with puromycin (1μg/ml).

14 Supplementary dataset S1. Expression of AXL, GAS6, Vimentin and E-Cadherin in 644 human cancer cell line using RNAseq technology. Supplementary dataset S2. Kinome profile with 1000nM R428. Supplementary dataset S3. Tumor cell line profile of docetaxel in combination with R428. Supplemental movie 1. Timelapse movie of HeLa cells treated with DMSO. Supplemental movie 2. Timelapse movie of HeLa cells treated with R428 (1μM). Supplemental movie 3. Timelapse movie of HeLa cells treated with docetaxel (3nM). Supplemental movie 4. Timelapse movie of HeLa cells treated with docetaxel (3nM) in combination with R428 (1μM).


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