Supplementary Material s20

Supplementary Material

The PI3 Kinase/mTOR Blocker NVP-BEZ235 Overrides Resistance against

Irreversible ErbB Inhibitors in Breast Cancer Cells

Caroline Brünner-Kubath1*, Waheed Shabbir1*, Victoria Saferding1, Renate Wagner1, Christian F.

Singer2, Peter Valent3,6, Walter Berger4, Brigitte Marian4, Christoph C. Zielinski5,6, Michael

Grusch4, Thomas W. Grunt1,6

1Signaling Networks Program, Division of Oncology, Department of Medicine I; 2Department of

Obstetrics/Gynecology; 3Division of Hematology/Hemostaseology; 4Institute of Cancer Research;

5Division of Oncology, Department of Medicine I, Medical University Vienna; 6Ludwig Boltzmann Cluster

Oncology, Vienna, Austria.

*These authors contributed equally to the study

Corresponding author: Dr. Thomas W. Grunt

Ludwig Boltzmann Cluster Oncology & Division of Oncology

Department of Medicine I, Medical University Vienna

Waehringer Guertel 18-20, A-1090 Vienna, Austria

Phone: +43-1-40400-5487 (-4430, -4429)

Fax: +43-1-40400-5465

E-mail: [email protected] Supplementary Table S1 Specification of the human neoplastic cell lines used in this study

Source Medium Subculture Markersa Breast Cancer ErbB1b ErbB2b ErbB3b ErbB4b ERc RARd RARe RARf FASNg BT20 ATCC RPMI 1640 1/10 weekly ++++ ++ +++ ? - ? ? ? +++ BT474 ATCC -MEM 1/3 weekly ++ +++++ ++++ + + +++ - + +++ MCF7 ATCC DMEM 1/10 weekly + + ++ + ++++ ++++ - ++ ++ MDA231 ATCC -MEM 1/12 weekly +++ ++ + + ? ? ? ? ? MDA361 ATCC DMEM 1/3 weekly + ++++ -/+ + ++ +++ - + +++ MDA453 ATCC -MEM 1/6 weekly - +++ ++ ++ - + - - ++ SKBR3 ATCC DMEM 1/8 weekly +++ +++++ ++ - - ++ - + ++++ T47D ATCC DMEM 1/10 weekly + ++ +++ +++ ++ + ++ ? ++ ZR7530 ATCC -MEM 1/2 weekly ? +++++ ? ? - ? ? ? ? Ovarian Cancer A2774 M. Krainerh -MEM 1/10 every 3d ++ - ? ? ? ? ? ? + A2780 M. Krainerh RPMI 1640 1/15 every 3d + + + + - +++ ++ ++ ++ A2780ADR M. Krainerh RPMI 1640 1/15 every 3d ++ ++ + ++ - +++ + + +++ CAOV3 ATCC DMEM 1/15 weekly ++ -/+ ? ? ++ ++ - + ++ H134 H.J. Broxtermani -MEM 1/12 weekly ++ + ? ? ? + - ++ ++ HEY R.N. Buickj DMEM 1/20 every 3d ++ + ? ? ? ++ - ++ ++ HOC-7 R.N. Buickj -MEM 1/20 every 3d + + ? ? ? + - ++ + OVCAR-3 ATCC -MEM 1/10 weekly ++ + ? ? + ++ - +++ ++ PA-1 ATCC -MEM 1/5 weekly ++ ++ ? ? ? + - ++ ++ SKOV3 ATCC -MEM 1/10 weekly ++ +++++ ? ? - ++ - ++ + TR-170 B.T. Hillk -MEM 1/8 weekly - ++ ? ? ? ++ - ++ + Leukemia CEM U. Jaegerh RPMI 1640 1/5 every 3d ------/+ ? ? ?

a- negative, + weakly positive, ++ moderately positive, +++ positive, ++++ strongly positive, +++++ overexpressed bErbB1 (EGF receptor/HER1), ErbB2 (HER2/Neu), ErbB3 (HER3), ErbB4 (HER4) (determined by Western blot analysis) cestrogen receptor-alpha (determined by enzyme immunoassay) dretinoic acid receptor-alpha (determined by reverse transcription-PCR) eretinoic acid receptor-beta (determined by reverse transcription-PCR) fretinoic acid receptor-gamma (determined by reverse transcription-PCR) gfatty acid synthase (determined by Western blot analysis) hMed. Univ. Vienna, Austria iFree Univ. Hospital, Amsterdam, The Netherlands jUniv. Toronto, ON kImperial Cancer Res. Fund, London, UK Supplementary Table S2 Specification of the primary and secondary antibodies used for Western blotting

Primary Antibodies Specificity Type Dilution Supplier

ErbB1 rabbit polyclonal IgG 1:500 Cell Signaling Technology, Boston, MA pErbB1(Tyr1068) rabbit polyclonal IgG 1:1,000 Cell Signaling Technology, Boston, MA ErbB2 rabbit polyclonal IgG 1:200 Santa Cruz Biotechnology, Santa Cruz, CA pErbB2(Tyr1248) rabbit polyclonal IgG 1:200 Santa Cruz Biotechnology, Santa Cruz, CA ErbB3 rabbit monoclonal IgG 1:1,000 Cell Signaling Technology, Boston, MA pErbB3(Tyr1289) rabbit polyclonal IgG 1:1,000 Cell Signaling Technology, Boston, MA ErbB4 rabbit monoclonal IgG 1:1,000 Cell Signaling Technology, Boston, MA pErbB4(Tyr1284) rabbit monoclonal IgG 1:1,000 Cell Signaling Technology, Boston, MA MEK1/2 rabbit polyclonal IgG 1:1,000 Cell Signaling Technology, Boston, MA pMEK1/2(Ser217/221) rabbit polyclonal IgG 1:1,000 Cell Signaling Technology, Boston, MA ERK1,2 rabbit polyclonal IgG 1:3,000 Upstate Biotechnology, Lake Placid, NY pERK1/2 rabbit polyclonal IgG 1:1,000 Cell Signaling Technology, Boston, MA PTEN rabbit monoclonal IgG 1:1,000 Cell Signaling Technology, Boston, MA AKT rabbit polyclonal IgG 1:1,000 Cell Signaling Technology, Boston, MA pAKT(Ser473) rabbit polyclonal IgG 1:1,000 Cell Signaling Technology, Boston, MA pAKT(Thr308) rabbit polyclonal IgG 1:1,000 Cell Signaling Technology, Boston, MA pGSK3ß(Ser9) rabbit polyclonal IgG 1:1,000 Cell Signaling Technology, Boston, MA mTOR rabbit polyclonal IgG 1:1,000 Cell Signaling Technology, Boston, MA pmTOR(Ser2448) rabbit polyclonal IgG 1:1,000 Cell Signaling Technology, Boston, MA pS6(Ser240/244) rabbit polyclonal IgG 1:2,000 Cell Signaling Technology, Boston, MA / tubulin rabbit polyclonal IgG 1:1,000 Cell Signaling Technology, Boston, MA Actin goat polyclonal IgG 1:200 Santa Cruz Biotechnology, Santa Cruz, CA

Secondary Antibodies Specificity Source Label Dilution Supplier

Rabbit IgG Donkey Peroxidase 1:15,000 Promega, Madison, WI Goat IgG Donkey Peroxidase 1:15,000 Santa Cruz Biotechnology, Santa Cruz, CA Rabbit IgG Goat Peroxidase 1:2,000 Cell Signaling Technology, Boston, MA Supplementary Figure Legends

Supplementary Fig. S1 The differential sensitivity of 10 breast (a-d) and 3 ovarian (e, f) cancer cell lines against the in vitro growth inhibitory action of the irreversible small molecular ErbB1/2 blocking drug pelitinib (EKB-569) (a, b, e, f) and the irreversible ErbB1/2/4 inhibitor canertinib (CI-1033) (c, d)

(0–20 µM, 72h) as determined by a formazan dye assay. (a, c, e) Dose response relationships. (b, d, f)

IC50-values determined from the dose response curves (details see Material and methods). ErbB- negative CEM leukemia cells (Grunt et al., 2007) were used as negative control cells. MeansSD, n3.

Supplementary Fig. S2 Pelitinib-refractory phosphorylation of AKT in pelitinib-resistant T47D cells is not caused by activation of AKT via the IGF-IR pathway. a Cells were exposed to pelitinib alone or together with the IGF-IR inhibitor PPP. The levels of pAKT remained high in pelitinib-treated cells irrespective of the presence of PPP as demonstrated by Western blotting. Actin was used as loading control. b T47D cells were exposed to 0.01–10 µM PPP. Seventy two hours later the antiproliferative activity of PPP was determined using a formazan dye assay. MeansSD, n3.

Supplementary Fig. S3 The dose-dependent effects of the dual PI3K/mTOR inhibitor NVP-BEZ235, the mTOR inhibitor rapamycin, and the AKT1/2 inhibitor Akti-1/2 on expression/phosphorylation of AKT,

S6 and ERK1,2 in ErbB drug-sensitive SK-BR-3 and -resistant T47D cells as demonstrated by Western blot analysis. Actin was used as loading control.

Supplementary Fig. S4 The dose-dependent effects of the MEK1/2 inhibitors AZD6244 and U0126 on expression/phosphorylation of MEK1,2 and ERK1,2 in ErbB drug-sensitive SK-BR-3 and -resistant

T47D cells as demonstrated by Western blot analysis. α,β-tubulin was used as loading control. Brünner-Kubath et al.

CEM MDA231 ZR7530 T47D SW527 SKBR3 A MCF7 BT474 MDA453 B BT20 MDA361 17 110 16

) 15 l 100 o 14 r t )

n 90 13 o

C 80 µ

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50 i b 7 n i t m 40 i 6 l u

30 P l l 4 e 20 3 C 10 2 1 0 0 0 2 4 6 8 10 12 14 16 18 20 Pelitinib (µM)

C SKBR3 T47D D

110 14 13 )

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E HOC7 SKOV3 CAOV3 F 14 110 13

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5 8 % 60 C I (

7 r b i e 50 6 n b i t i m 40 l 5 u e 4 P N 30

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Supplementary Figure S1 Brünner-Kubath et al.

120 ) 6h Pelitinib (µM) - 4 8 l

o 110 r t 100 2µM, 6h PPP - - + - + n o 90 C

pAKT(Ser473) f 80 o 70 % (

60 AKT r e 50 b 40 m

Actin u 30 N

e 10 C 0 0,01 0,03 0,1 0,3 1 3 10 PPP (礛 )

SKBR3 T47D 6h NVP-BEZ235 (µM) 0 .01 .025 .05 .075 .1 .5 1 0 .01 .025 .05 .075 .1 .5 1 pAKT(Ser473) pAKT(Thr308) AKT pS6 S6 pERK1,2 ERK1,2 Actin

SKBR3 T47D 6h Rapamycin (µM) 0 .1 .25 .5 .75 1 5 10 0 .1 .25 .5 .75 1 5 10 pAKT(Ser473) pAKT(Thr308) AKT pS6 S6 pERK1,2 ERK1,2 Actin

SKBR3 T47D 6h Akti-1/2 (µM) 0 .01 .05 .1 .5 .75 1 2.5 5 0 .01 .05 .1 .5 .75 1 2.5 5 pAKT(Ser473) pAKT(Thr308) AKT pS6 S6 pERK1,2 ERK1,2 Actin

SKBR3 T47D 6h AZD6244 (µM) 0 0.1 1 5 10 20 0 0.1 1 5 10 20 pMEK1,2 MEK1,2 pERK1,2 ERK1,2 ,-Tubulin

SKBR3 T47D 6h U0126 (µM) 0 10 20 40 80 0 10 20 40 80 pMEK1,2 MEK1,2 pERK1,2 ERK1,2 ,-Tubulin

Supplementary Figure S4