Supplemental Materials and Methods s5

SUPPLEMENTAL MATERIALS AND METHODS

Cell culture: T47D:A18/neo and T47D:A18/PKCα cells were maintained in RPMI 1640 with 10% FBS, non-essential amino acids (1X), 6 ng/ml insulin and G418 (500 g/ml).

Charcoal-stripped serum was used when indicated. T47D:C42 cells were grown in phenol-red free RPMI 1640 with 10% charcoal-stripped FBS, non-essential amino acids

(1X) and 6 ng/ml insulin. MCF-7/Tam-S and MCF-7/Tam-R cells were maintained in

DMEM with 10% FBS and in DMEM with 10% charcoal-stripped FBS, respectively. To generate stable cell lines overexpressing active Notch-4 (Notch-4IC), T47D:A18neo cells were transfected in serum-free medium using the X-tremeGENE HP DNA Transfection

Reagent (Roche, Cat. No. 06 366 236 001) with plasmid pCDNA3-Notch4IC together with pBABE-puro (Addgene, Cat. No. 1764). Following incubation for 3 days in complete medium, the cells were treated with 1 µg/ml of Puromycin in complete medium. After one month of selection, resistant clones were pooled and the resulting transformed cell line (T47D:A18/N4IC) was tested for exogenous Notch-4IC expression.

In parallel, a cell line containing the corresponding empty vector (pCDNA3) plus pBABE-puro was similarly generated as a control (T47D:A18/puro).

Plasmids: Notch-4IC in pcDNA3.0 was a gift from Brian J. Nickoloff (Loyola

University, Chicago, IL).

Antibodies and chemicals: Antibodies to Notch-1 (C-20), Notch-4 extracellular (L-16),

Notch-4 C-terminal (H-225), PKCα (C-20), c-Jun (D), c-Fos (K-25), normal rabbit IgG, and goat IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Notch-4

C-terminal antibody LSBio-B3498 was from Lifespan Biosciences (Seattle, WA), and

HEY-1 antibody and GAPDH antibody from Chemicon Internation (Temecula, CA).

1 Notch-4 antibodies were validated using siRNA to Notch-4 for L-16 and transfection of

Notch-4IC for H-225 and LSBio-B3498. L-16 was used for immunohistochemistry as previously described, as it generated the cleanest background. H-225 was used for

Western blotting unless otherwise stated. The most abundant validated band (64 kDa) is shown in Western blots. Secondary antibodies anti-goat, anti-mouse and anti-rabbit (IgG-

HRP) were purchased from Vector Laboratories (Burlingame, CA), Santa Cruz

Biotechnology and Amersham Biosciences (Piscataway, NJ), respectively. Orally active

γ-secretase inhibitor (MRK-003) was a kind gift of Merck Research Laboratories,

Boston, MA. Tamoxifen (T5648), Estradiol (E8875), Tween 80 (P8074), PEG400

(P3265), and carboxymethylcellulose (C9481) were purchased from Sigma (St, Louis,

Real-time RT-PCR: Total RNA was isolated from cells or tumors with RNeasy Mini Kit according to the manufacturer's instructions (Qiagen, CA). Total RNA was also isolated from archival clinical samples: 4µm formalin-fixed, paraffin-embedded (FFPE) sections from 20 cases of breast cancers were processed using the RNeasy FFPE kit (Qiagen,

Valencia, CA) according to the manufacturer’s instructions. Total RNA was treated with

RQ1 RNase-free DNase (Promega, Madison, WI) to remove genomic DNA.

RNA Isolation, Affymetrix GeneChip Human Gene 1.0 ST Array, microarray analysis and bio-informatics. We analyzed the gene expression profiles of T47D:A18 parental cells, T47:A18/Notch-4IC, T47D:A18/Notch-1IC and T47D:A18/PKCα cells.

Briefly, cells were grown in complete medium or charcoal-stripped (estrogen-depleted) medium and RNA was isolated as described above. Three biological replicates were analyzed for each experimental condition. Expression profile analysis was performed at

2 the Genomics Core Facility at UMC using Affymetrix GeneChip® Human Gene 1.0 ST

Arrays. Procedures for cDNA synthesis, labeling and hybridization were carried out according to the manufacturer's protocol (www.affymetrix.com). Expression of selected transcripts was validated by Q-RT-PCR. Gene-level expression values were derived from CEL files by quantile sketch normalization using the model-based Robust Multichip

Average (RMA) method.3 The GenePattern software hosted at the Broad Institute was used to generate gene expression heat maps.4 We selected transcripts from

T47D:A18/Notch-1IC and T47D:A18/Notch-4IC cells with expression values showing a log2 ratio ≥ 1.5 and ≤ -1.5 (p ≤ 0.0005) compared to the T47D:A18/neo line. Using this

3 subset, a pairwise complete-linkage hierarchical clustering of normalized log2 values from T47D:A18/neo, T47D:A18/Notch-1IC, T47D:A18/Notch4IC and T47D:A18/PKC cell lines, with Pearson correlation for column and row distance measuring, was used to generate clusters. The clusters were displayed in a heat map format where values were converted to colors using the mean, minimum, and maximum values in the dataset

(Global coloring scheme). The largest values are displayed as yellow, the smallest values as cyan, and intermediate values as black.

Transient overexpression of Notch-4IC in T47D:A18 cells: To validate the Notch-4IC band observed in the Western blot, T47D:A18 cells were transfected with different concentrations of pCDNA3 Notch-4IC plasmid (0.062, 0.125, 0.25, 0.5 or 1µg/ml). After

48 hours cells were harvested and cell lysates were prepared. Proteins were separated by

SDS-PAGE electrophoresis and Notch-4 protein was detected using anti-Notch-4 H225

(sc-5594, Santa Cruz Biotechnology Inc.) antibody.

3 Effect of MRK003 on Notch-4 cleavage in T47D:A18/PKCα cells. T47D:A18/PKCα cells expressing high levels of endogenous Notch-4 were cultured in RPMI-Phenol red containing medium supplemented with 10% Fetal Bovine Serum (FBS) for 4 days, and then treated with 5μM MRK003 for 24h. After the treatment cells were harvested, lysed and analyzed by Western blotting using two different Notch-4 antibodies: H-225 (sc-

5594, Santa Cruz Biotechnology Inc.) or LS-B3498 (LifeSpan BioSciences, Inc.).

Quantification of the band intensity was performed using the ImageJ software freely available at http://rsb.info.nih.gov/ij/. Values were normalized by β-actin.

Xenograft tumors: T47D:A18/PKCα cells were grown in RPMI1640 with 10% FBS, non-essential amino acid (1X) and 6 ng/ml insulin containing G418 (500 μg/ml), and

7 incubated at 37 °C and 5% CO2. Cells were washed two times in sterile PBS. 1×10 cells suspended in 100 μl of sterile PBS were inoculated bilaterally into the mammary fat pad on each side of mice. Under these conditions, palpable tumors developed in 13 days. On the 14th day, tumors were measured using a Vernier caliper. Average tumor volume was

0.28 ± 0.05 cm3. Animals were randomized into control and test groups (n = 8). Drug treatment was started: control (vehicle), TAM (1.5 mg TAM), GSI (MRK003 100mg/kg),

TAM plus GSI (1.5 mg TAM and 100mg/kg MRK003). TAM was administered by gavage five days a week whereas MRK003 was administered by gavage daily for the first three days of each weekly dosing cycle. (3 days ON, then 4 days OFF). Tumor volume

[(length x width2)/2] was monitored twice a week by Vernier caliper measurements for up to 4 weeks. Animals were sacrificed by CO2 asphyxiation. Tumors and small intestines were dissected and histopathological analysis was performed.

4 SUPPLEMENTAL FIGURE LEGENDS

Supplemental Figure 1: (A, B) Notch-1IC band validation. T47D:A18/PKCα cells were cultured in the presence of vehicle, 5μM DAPT or 5μM MRK003 for 24h. Lysates were prepared and proteins separated on SDS–PAGE and immunoblotted with anti-Notch-1 antibodies. (A) Western blot showing expression of Notch-1 (Transmembrane and cleaved form) after GSIs treatment using anti-Notch-1 (C20) antibody (Santa Cruz

Biotechnology) (left panel) and the cleaved Notch1 (Val1744) (Cell Signaling). Near- infrared fluorescence detection quantified by an Odyssey scanner allows simultaneous staining with 2 antibodies. Two-color simultaneous detection of Notch-1 (C20) and cleaved Notch1 (Val1744) bands were performed. IRDye 800CW secondary antibodies were used. Green fluorescence and red fluorescence was used for anti-Notch-1 (C20) and cleaved Notch1 (Val1744) antibodies, respectively. The left panel shows the green signal

(C20), which we routinely use to quantify Notch-1, while the right panel shows the merged dual-color image. Green bands react with C20 antibody alone, red bands react with the Val1744 antibody alone but not C20 (and therefore represent non-specific bands, since the C20 antibody is a polyclonal IgG raised against the C-terminal domain of

Notch-1). The yellow band recognized by both antibodies and indicated by the arrows was considered Notch1IC, because: 1) it reacts with both the Val1774 and C20 antibodies; 2) it has the expected molecular mass of ~96 kDa and 3) it is decreased by

GSI MRK003, and to a lesser extent by DAPT. Note that the effect of GSIs cannot be

5 adequately quantified from this image because of spectral overlap between the green and red signals. Thus, the effect of GSIs is likely to be underestimated in this image. We have previously reported1 that treatment with GSI in T47D cells decreases both the Notch1IC and Notch1TM bands, most likely due to Notch1 autoregulation, which we have observed in patients treated with GSI MK0752.5 (B) Densitometric scan from the dual-color

Western blot, identifying the effect of GSIs on the 96 kDa band recognized by both antibodies. Quantification of the band intensity was performed using the ImageJ software freely available at http://rsb.info.nih.gov/ij/. Values were normalized by β-actin and expressed as a ratio between the GSI treated/vehicle treated cells. (C) Identification of

Notch-4IC bands. T47D:A18 cells were transfected with increasing amounts of pCDNA-

3 expressing Notch-4IC. DNA amount was kept constant by adding empty pCDNA3.

Cells were lysed 48 hours after transfection and Western blotting was performed with

Notch-4 antibody H-225 (sc-5594; Santa Cruz Biotechnology, Inc.). (D) Notch-4 expression in T47D:A18 stable cell lines. Notch-4 expression was analyzed in the stable

T47D:A18 cell lines transfected with empty vector (T47:A18), active Notch-4 (Notch-

4IC) or PKCα. T47D:A18/Notch-4 cell were generated after transfection of T47D:A18 neo cells with the plasmid pCDNA3-Notch4IC together with pBABE-puro (Addgene,

Cat. No. 1764). Control cells were transfected with empty pCDNA3 and pBABE-puro.

Following incubation for 3 days in complete medium, the cells were treated with 1 µg/ml of Puromycin in complete medium. After one month of selection, resistant clones were pooled and the resulting transformed cell line (T47D:A18/N4IC) were tested for

Notch4IC expression using Notch-4 antibody (H225). In the figure non-contiguous lanes detected with identical settings were spliced together for comparison. (E) Notch-4 siRNA

6 selectively knocks down the 64 kDa band. 150 pmoles of Notch4 siRNA (sc-40137) and two control siRNAs (sc-44239 and sc-44236) were transfected in 2 x 105

T47D:A18/PKCa cells using the X-tremeGENE siRNA Transfection Reagent (Roche, cat

# 04 476 093 001). Proteins were extracted 48h post transfection and analyzed by

Western blot with the Notch4 antibody H-225 (sc-5594). (F, G, H) Effect of MRK003-

GSI on Notch-4 protein levels. T47D:A18/PKCα cells were cultured in the presence of

5μM MRK003 for 24h. Lysates were prepared and proteins separated on SDS–PAGE and immunoblotted with polyclonal anti-Notch-4 antibody H-225 (sc-5594; Santa Cruz

Biotechnology, Inc.) (F) or LS-B3498 (LifeSpan BioSciences, Inc.) (G). (H): densitometry of the 64 kDa band in panel C (left) and D (right). Quantification of band intensity was performed using the ImageJ software freely available at http://rsb.info.nih.gov/ij/. The molecular weight (MW) of the bands identified as Notch-4 protein were calculated using log(MW) and Rf (relative migration distance) based on prestained standards. Both antibodies showed that MRK003 drastically decreased the 64 kDa band. (I, J) Differential effects of DAPT and MRK003-Gamma Secretase Inhibitors on Notch-4 expression. T47D:A18/PKCα cells were cultured in complete medium (RPMI

1640 plus 10%FBS) and treated with 5μM of DAPT or MRK003 for 24h. Lysates were prepared and proteins separated on SDS–PAGE and immunoblotted with polyclonal anti-

Notch-4 antibody H-225 (sc-5594; Santa Cruz Biotechnology, Inc.). Panel I shows that the 64 kDa Notch-4IC band was increased by DAPT and decreased by MRK003. Panel J shows the quantification of band intensity performed using the ImageJ software freely available at http://rsb.info.nih.gov/ij/. Values were normalized by β-actin and expressed as a fold change between the Notch-4 band and β-actin.

7 Supplemental Figure 2: MRK003 GSI and TAM in combination decrease

T47D:A18/PKCα ERα-positive breast cancer xenograft growth. Palpable tumors developed in 14 days. Average tumor volume was 0.1 cm3. Animals were randomized into control and test groups (n=9). On the 17th day, drug treatment was started: control

(vehicle), TAM (0.5 mg), GSI (MRK-003 100 mg/kg), TAM/GSI (0.5 mg of TAM and

100 mg/kg of MRK-003). TAM was administered by gavage five days a week whereas

MRK-003 was administered by gavage for 3 days ON, then 4 days OFF (intermittent dose schedule, in order to minimize intestinal toxicity). Tumors were measured using a

Vernier caliper twice a week. Arrow marks the start of treatment.

Supplemental Figure 3: Tamoxifen reduces the toxicity of GSI MRK003 in estrogen- receiving mice. Mouse survival rates over the duration of treatment in the experiment described in Supplemental Figure 2. Arrow indicates the beginning of treatment. 1 mouse died in the vehicle group and 2 in the TAM alone group during the 55-day treatment. All but 3 mice in the GSI alone group died with severe diarrhea. No mice died in the

TAM/GSI group and no diarrhea was observed.

Supplemental Figure 4: MRK003 GSI alone and in combination with TAM caused cell death in xenografts after 1 week treatment. Histology (H&E) of T47D:A18 xenografts (x400) showed GSI, either alone or with TAM, caused cell death with nuclear

8 pyknosis (blue arrows), edema (yellow arrows), and hemorrhage (red arrows). Note that the combination has a more severe phenotype than either drug alone.

Supplemental Figure 5: TAM overcomes the intestinal toxicity of MRK003.

Histology of small intestine after 1 week treatment showed GSI alone caused epithelial erosion and dilation of crypts while GSI with TAM restored normal intestinal architecture of crypts.

Supplemental Figure 6. MCF-7/Tam-R cells overexpress Notch-4IC and Notch-4 knockdown inhibits proliferation and reverses the TAM-resistant phenotype. (A)

Western Blot analysis using antibody H-225 showing upregulation of Notch-4IC in

MCF-7 Tamoxifen resistant cells (MCF-7/Tam R). (B) Cell growth assays after silencing

Notch-4 in MCF-7/Tam-R. MCF-7/Tam-R cells transfected with scrambled shRNA

(SCB) or Notch-4 shRNA (shNotch-4) were cultured in the presence or absence of 4- hydroxytamoxifen (4-OH TAM, 1μM). Cell growth was measured by Crystal Violet

Assay for the indicated times. Results represent the mean ± s.d. for three independent samples.

Supplemental Table 1: Effect on T47D:A18/PKCα cell growth upon treatment with different doses of GSI MRK003, 4 OH-TAM and combined treatment, tabulated as

Fraction Affected and derived Combination Index.

9 Supplemental Table 2: Microarray analysis. Cell-cycle-related genes up-or down- regulated in charcoal-stripped (estrogen-depleted) medium in T47D:A18/Notch-4IC,

T47D:A18/Notch-1IC, T47D:A18/PKCα compared to parental T47D:A18 cells.

Expression ratios between complete medium and charcoal-stripped medium (CM/CS) are also shown.

Supplemental Table 3: Microarray analysis. Type I interferon-related genes up- regulated in T47D:Notch4IC and T47D:PKCα cells compared to parental T47D:A18 cells. Expression ratios in estrogen-depleted and complete media are shown.

T47D:A18/Notch-1IC cells are shown for comparison. Expression ratios between complete medium and charcoal-stripped medium (CM/CS) are also shown.

References

1 Rizzo P, Miao H, D'Souza G, Osipo C, Song LL, Yun J et al. Cross-talk between

notch and the estrogen receptor in breast cancer suggests novel therapeutic

approaches. Cancer Res 2008; 68: 5226-5235.

10 2 Yao K, Rizzo P, Rajan P, Albain K, Rychlik K, Shah S et al., Notch-1 and notch-4

receptors as prognostic markers in breast cancer. Int J Surg Pathol 2011; 19: 607-

3 Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U et al.,

Speed TP, Exploration, normalization, and summaries of high density

oligonucleotide array probe level data. Biostatistics 2003; 4: 249-264.

4 Eisen MB, Spellman PT, Brown PO, Botstein D, Cluster analysis and display of

genome-wide expression patterns. Proc Natl Acad Sci U S A 1998; 95: 14863-14868.

5 Albain K, , Czerlanis C, Zlobin A, Covington KR, Rajan P, Godellas C, et al. ,

Modulation of Cancer Stem Cell Biomarkers by the Notch Inhibitor MK0752 Added

to Endocrine Therapy for Early Stage ER+ Breast Cancer. Cancer Res 2011; 71: 97s.