Identification of Cell Surface and Secreted Proteins Essential For

Identiﬁcation of cell surface and secreted proteins essential for tumor cell survival using a genetic suppressor element screen

Marina S Gelman1, X Katherine Ye1, Robert Stull1, David Suhy1, Liang Jin1, Dean Ng1, Bruce Than1, May Ji1, Alison Pan1, Paul Perez1, Yan Sun1, Patricia Yeung1, Luz Maria Garcia1, Rachel Harte1, Yan Lu1, Elizabeth Lamar1, Roya Tavassoli1, Scot Kennedy1, Stephen Osborn1, Daniel J Chin1, Kay Meshaw2, Tatyana A Holzmayer1,3, Sergey A Axenovich*,1 and Arie Abo*,1

1PPD Discovery, Inc., 1505 O’Brien Drive, Menlo Park, CA 94025, USA; 2Piedmont Research Center, PPD Discovery, Inc., 3300 Gateway Centre, Morrisville, NC 27560, USA

Survival factors play critical roles in regulating cell growth Cell surface receptors and soluble growth factors are in normal and cancer cells. We designed a genetic screen to amenable to monoclonal antibody therapeutics (mAbs). identify survival factors which protect tumor cells from The mAb technology (Kohler and Milstein, 1975) has apoptosis. A retroviral expression library of random cDNA resulted in a number of approved antibody treatments fragments was constructed from cancer cells and used to for cancer (Carter, 2001), including trastuzumab (Her- transduce the colon carcinoma cell line HCT116. Recipient ceptins), a humanized mAb against the receptor cells were functionally selected for induction of caspase 3- tyrosine kinase ErbB2, for ErbB2-overexpressing meta- mediated apoptosis. Analyses of over 10 000 putative static breast cancer (Cobleigh et al., 1999) and genetic suppression elements (GSEs) sequences revealed bevacizumab (Avastins), a humanized mAb against cognate gene candidates that are implicated in apoptosis. the vascular endothelial growth factor, for treatment of We further analysed 26 genes encoding cell surface and metastatic colorectal cancer (Kabbinavar et al., 2003). secreted proteins that can potentially serve as targets for In addition to complement-mediated cell lysis, the therapeutic antibodies. Tetracycline-inducible GSEs from mechanism of action of Herceptins, as well as anti- several gene candidates induced apoptosis in stable HCT epidermal growth factor receptor (EGFR) antibody 116 cell lines. Similar phenotypes were caused by RNAi (Yang et al., 1999), includes interference with growth derived from the same genes. Our data suggest requirement factors binding to their receptors and acceleration of for the cell surface targets IGF2R, L1CAM and SLC31A1 receptor internalization thereby inhibiting cancer pro- in tumor cell growth in vitro, and suggests that IGF2R is gression. Some anticancer mAbs promote apoptosis required for xenograft tumor growth in a mouse model. either in cancer cells or in proliferating endothelial cells Oncogene (2004) 23, 8158–8170. doi:10.1038/sj.onc.1208054 leading to impaired tumor blood supply. For example, Published online 13 September 2004 rituximab (Rituxans), a mAb against CD20, has been approved for treatment of B-cell non-Hodgkin’s lym- Keywords: genetic suppressor element (GSE); cancer phoma (Byrd et al., 2002), and Vitaxint, a mAb against therapeutic target; tumor cell; apoptosis; essential gene; integrin avb3, is currently in Phase I/II clinical trials xenograft (Gutheil et al., 2000; Tucker, 2003). The ability to identify cellular targets, which protect tumor cells from apoptosis, could lead to a more focused development of anticancer therapeutics. Introduction To identify such cell surface and secreted targets, we have used the GSXt System. The GSXt technology ONCOGENOMICS The fine tuning of signals controlling cell growth and allows isolation of genetic suppression elements (GSEs), programmed cell death (apoptosis) is essential for cell 100–500 bp biologically active cDNA fragments that survival (Hanahan and Weinberg, 2000). In cancer cells, interfere with the function of their cognate genes this signaling is unbalanced due to overexpression or (Roninson et al., 1995; Gudkov and Roninson, 1997). mutation of oncogenes and/or antiapoptotic survival GSXt functional screens were previously used to factors. Inhibition of these molecular targets offers a identify viral genes that play an important role in the therapeutic opportunity for cancer treatment. infectious cycle of bacteriophage l (Holzmayer et al., 1992) or human immunodeficiency virus (Dunn et al., *Correspondence: A Abo, 1018 4th Av., Suite 119, Oakland, CA 1999), to clone novel tumor suppressor genes (Garkavt- 94606, USA; E-mail: [email protected]; and sev et al., 1998), genes involved in apoptotic response SA Axenovich; E-mail: [email protected] 3Dr Holzmayer died on February 27, 2002 (Neznanov et al., 2003), genes controlling yeast pher- Received 2 February 2004; revised 23 March 2004; accepted 24 July omone response pathway (Caponigro et al., 1998) and 2004; published online 13 September 2004 genes that modulate cellular response to chemotherapy Cell surface and secreted cancer targets MS Gelman et al 8159 (Gudkov et al., 1994; Gallagher et al., 1997; Levenson Results et al., 2000; Gros et al., 2003). Most recently, using Apoptosis GSE screen design GSXt strategy, a large set of genes involved in proliferation of breast carcinoma cells has been identi- We have developed a GSE screen to identify genes that fied (Primiano et al., 2003). regulate cancer cell survival and apoptosis. As illu- Here, we describe a functional screen for GSEs strated in Figure 1a, the screen was designed to survey capable of inducing apoptosis in cancer cells. We have the complete cancer cell transcriptomes to select genetic focused on the validation of genes corresponding to cell suppressors that activate caspase 3. The screen was surface and secreted proteins. Using both stable cell conducted in HCT116 colon carcinoma cells for the lines with inducible GSE expression and RNAi transfec- following reasons: (i) high sensitivity to apoptotic tion, we demonstrate the role of several membrane and stimuli, (ii) amenability to high-throughput retrovirus- secretory proteins in proliferation and survival of colon mediated gene transfer and (iii) availability of somatic carcinoma cells. gene knockout derivatives (Torrance et al., 2001). To

Figure 1 Apoptosis GSE screen. (a) Apoptosis GSE screen schematic. 1. Random fragment normalized (equal abundance) cDNA library was prepared from pooled total RNA of eight cancer cell lines and subcloned into IPTG-inducible retroviral vector V99 in an antisense orientation (see panel b). 2. The library was transfected into Phoenix-Eco retrovirus packaging cells to generate viral particles encoding random cDNA fragments. 3. HCT-116 cells were transduced with the viral supernatant. 4. Expression of random cDNA fragments was induced with 50 mM IPTG and floating cells were collected at 0, 24, 48 and 72 h after IPTG induction. Floating cells positive for active caspase-3 were collected. 5. Genomic DNA was isolated from collected cells. Putative GSEs were recovered by PCR and recloned in the V99 vector. 6. The screen was repeated for three iterations. 7. About 10 000 putative GSE clones were sequenced and analysed. (b) Expression vector design. V99 is a modified MoMLV vector containing the coding sequence for a truncated murine CD8a0 protein, which serves as a marker of viral transduction. An internal CMV promoter, modified by the addition of three lac operons, permits IPTG inducible expression of the random fragment library. The cloning cassette is flanked by rare-restriction sites, sequencing primer sites, unique PCR priming sites and a three-frame TGA sequence. (c) Active caspase 3 antibody staining of attached (right panel) and floating (left panel) HCT116 cells transduced with the GSE library at 24 h post IPTG induction during the second iteration of the screen. (d) Number of GSE screen hits per gene distribution after the third iteration of the screen

Oncogene Cell surface and secreted cancer targets MS Gelman et al 8160

Figure 1 Continued

achieve transcript diversity and minimize bias against empty vector exhibited caspase 3-dependent apoptosis differentially expressed genes, we constructed a library (data not shown). Approximately 1.5 Â 109, 1.9 Â 108 of approximately 40 million random cDNA fragments and 3.8 Â 107 cells transduced with GSE library were in retroviral expression vector (Figure 1b) from pooled passed through the first, second and third consecutive transcriptomes of eight tumor cell lines. In addition, rounds of selection for caspase 3-positive cells, tumor cell mRNAs were normalized for equal abun- respectively. After the third round of selection, over dance prior to random fragmentation to increase the 10 000 library clones were isolated and sequenced, and likelihood of isolating GSEs corresponding to rare subsequent GSE screen hits that induce apoptosis were messenger RNAs. To ensure selection of potent identified. cytotoxic GSEs, the library expression was controlled by an isopropyl-b-thio-galactoside (IPTG)-regulated Apoptogenic GSE screen hits derived from the genes CMV promoter (Chang and Roninson, 1996). We used encoding extracellular proteins the processing of caspase 3 zymogen as an apoptotic marker to select GSEs that initiate apoptotic pro- The isolated putative GSEs were derivatives of a gramme. Cells containing active caspase 3 were diverse group of approximately 950 genes. While the detected by antibody staining, flow sorted, and majority of GSE screen hit sequences corresponded to subsequent GSE screen hits were isolated from known mRNA sequences (74%), we also identified genomic DNA and sequenced. We have determined numerous putative GSE sequences found only in that induction of apoptosis by a variety of stimuli in genomic DNA and ESTGenbank sequences (26%). HCT116 cells causes more than 85% of apoptotic cells As shown in Figure 1d, in many cases, we identified a to float (data not shown). Therefore, to increase the single putative GSE per gene that are likely to throughput of the screen we selected caspase 3-positive represent nonspecific DNA fragments. High incidence cells from the floating cell population (Figure 1c). To of single GSE screen hits may also suggest that either capture GSEs with different expression kinetics, cells the screen was not saturated or certain transcripts were harvested at 0, 24, 48 and 72 h post IPTG were under represented in the library. Therefore, treatment. Approximately 5% of cells transduced with single GSE screen hits were excluded from the

Oncogene Cell surface and secreted cancer targets MS Gelman et al 8161 subsequent studies and cognate genes identified by two GSE target genes are expressed in tumor cell lines or more putative GSE sequences were considered as bona fide hits. Multiple GSE screen hits were enriched To confirm the expression of the putative GSE cognate for approximately 363 genes encoding cell surface genes in the GSE screen cell model, we performed receptors, signaling proteins, transcription factors mRNA expression analysis on HCT116 cell lines using and other proteins. Approximately 21% of the puta- custom Agilent oligonucleotide microarrays. For the 26 tive GSEs were derived from genes with unknown cognate genes, two different oligonucleotides were function. spotted in triplicate on each array. These oligonucleo- Since extracellular targets are amenable for thera- tides differed from one another in their complementary peutic antibody development, we focused our analyses region on the target mRNA, although both regions were on cell surface and secreted proteins. In total 25 genes located within terminal 1500 nucleotides of the target in this category were identified by two or more putative mRNA. All 26 loci showed expression in HCT116 cells, GSEs (Table 1). We have included in the study the albeit at low to moderate levels, indicating the presence insulin-like growth factor II receptor/mannose-6- of cognate transcripts for each of the putative GSEs phosphate receptor (IGF2R) gene (which yielded isolated in the apoptosis screen (Table 1). a single antisense putative GSE in our apoptosis The same custom Agilent oligonucleotide microarrays screen) since it was previously shown to promote were used to further compare the relative expression levels apoptosis in different cancer cell models (Souza et al., of five cognate genes, XPR1, FBN2, IGF2R, COL4A2 1999; Chen et al., 2002). and SLC31A1, in tumor cell lines SKOV3, PC3, A549, Typically, isolation of clusters of putative GSEs MDA-MB-231 and HCT116. Each of the five genes represented by overlapping sequences (termed multiple generated a detectable signal in each of the five tumor lines overlapping elements) is indicative of a strong selection (data not shown). Aside for the relatively strong expression within a particular region of the mRNA. Multiple of COL4A2 in SKOV3 cells, the expression profile of each overlapping elements were found for 12 out of 26 genes loci was quite similar among the five cell lines examined. (Table 1). Most of the isolated putative GSEs were We have assessed protein expression levels of three antisense elements, while some were found in a sense cognate genes, XPR1, IGF2R and COL4A2, in the same orientation (COL4A2 and CD81). panel of five tumor cell lines using immunocytometry

Table 1 List of the cell surface and secreted protein genes identiﬁed by two or more putative GSEs in the apoptosis screen Gene Accession Gene name Expression in Number of Putative GSE coordinates, 50 to 30 symbol HCT116a clones/ probe 1/probe 2 sequences

APLP2 XM_050724 Amyloid b (A4) precursor-like protein 2 0.33/0.32 3/3 733–191, 1109–879, 2158–1772 APP NM_000484 Amyloid b (A4) precursor protein 0.49/0.68 6/6 886–452, 822–546, 890–520, 899–556, 2140–1853, 2140–1857 ART4 NM_014062 ART-4 protein 0.46/0.63 2/2 343–40, 548–231 BST2 NM_004335 Bone marrow stromal cell antigen 2 0.11/0.26 8/8 65–10, 352–10, 349–15, 352–24, 346–54, 352–52, 335–83, 348–199 CD63 NM_001780 CD63 antigen (melanoma 1 antigen) 1.23/1.22 4/4 354–27, 614–184, 685–380, 747–417 CD81 NM_004356 CD81 antigen 0.74/1.16 3/3 557–286, 630–824, 635–824 COL4A2 XM_049904 Collagen, type IV, alpha 2 0.10/0.25 3/3 118–762, 1110–940, 1153–919 DBI M15887 Endozepine 0.66/0.81 4/3 164–5, 170–3, 207–3 ENSA XM_041911 Endosulﬁne alpha 0.09/0.12 2/2 342–1, 346–24 FBN2 NM_001999 Fibrillin 2 0.17/0.07 2/2 3196–2884, 3752–3481 FIBL-6 XM_053531 Fibulin 6 0.02/0.03 2/1 14087–13877 GFER NM_005262 Growth factor, erv1 0.71/2.87 3/3 1533–1405, 1687–1560, 1685–1568 (Saccharomyces cerevisiae) like HDGF NM_004494 Hepatoma-derived growth factor 0.42/0.82 4/4 707–431, 737–415, 699–575, 770–558 IGF2R NM_000876 Insulin-like growth factor 2 receptor 0.17/0.16 1/1 5179–4920 IK NM_006083 IK cytokine, downregulator of HLA II 0.11/0.16 3/3 298–71, 378–42, 413–48 ITGA3 NM_005501 Integrin, alpha 3 (antigen CD49C) 1.30/1.13 2/2 744–367, 749–474 L1CAM NM_000425 L1 cell adhesion molecule 1.61/0.37 2/1 1741–1529 NRP1 AF018956 Neuropilin 0.05/0.07 2/2 1028–621, 2298–2000 PDAP1 NM_014891 PDGF-associated protein (HASPP28) 0.39/0.45 5/5 136–20, 128–46, 322–24, 330–60, 317–141 PRNP NM_000311 Prion protein (p27–30) 0.65/0.52 2/2 481–333, 668–345 PTMA NM_002823 Prothymosin, alpha 3.57/3.56 2/2 290–139, 467–405 SILV BC001414 Silver (mouse homolog) like 0.06/0.03 3/2 353–12, 849–649 SLC31A1 NM_001859 Solute carrier family 31, member 1 0.10/0.19 2/2 36–1, 1389–1235 SLC5A6 NM_021095 Solute carrier family 5, member 6 0.18/0.06 3/2 698–583, 718–583 STC2 XM_037313 stanniocalcin 2 0.30/0.07 2/2 139–30, 188–74 XPR1 NM_004736 Xenotropic/polytropic retrovirus receptor 0.38/0.06 2/2 304–130, 2102–1772 aRelative mRNA expression determined by Agilent microarray hybridization. Average intensity of the signal for a speciﬁc oligonucleotide probe 1 and probe 2 was normalized to the average intensity of the signal from the two b-2-microglobulin (B2M) probes

Oncogene Cell surface and secreted cancer targets MS Gelman et al 8162 (data not shown). XPR1 protein expression was cells expressing ErbB2 antisense exhibited proliferation detected at low levels in all cell lines except A549. rates similar to vector control. However, in the IGF2R protein is expressed at a low level in all five cell SLC31A1 cell line, proliferation was inhibited by more lines. COL4A2 protein is expressed in low to moderate than 25% as early as 48 h post doxycycline induction levels in all cell lines with considerably higher expression (Table 3). SLC31A1 cells had plating efficiency similar in SKOV3 cells. to vector control cells as compared to other cell lines (Figure 2c), indicating a role for SLC31A1 in cell Apoptogenic GSEs inhibit tumor cell survival and proliferation. proliferation Furthermore, the plating efficiencies of SLC31A1 cells in soft agar is rather similar to vector control cells To determine whether expression of individual putative (Figure 2e), whereas IGF2R and ErbB2 antisense cells GSEs identified in the apoptosis screen can be deleter- exhibited very low plating efficiency. Interestingly, ious to cell survival and proliferation, we have generated several clones derived from IGF2R putative GSE cell stable cell lines with inducible expression of putative lines grew on soft agar independently of doxycycline GSEs corresponding to the cell surface adhesion protein treatment. In contrast, all other cell lines including L1CAM, IGF2R and a copper transporter SLC31A1. ErbB2 antisense cells demonstrated doxycycline-depen- As a positive control for this set of experiments, we dent decrease in colony number (Figure 2e). To correlate established a line expressing 1.5 kB c-ErbB2 cDNA colony size to GFP expression, the soft agar colonies fragment cloned in an antisense orientation into the were further examined under fluorescent microscope same construct in place of a GSE. This ErbB2 antisense (Figure 2f). We found that in the case of vector control, sequence was previously shown to downmodulate most large colonies were highly fluorescent after endogenous ErbB2 expression and inhibit anchorage- exposure to doxycycline. However, in GSE cell lines, independent growth of ovarian carcinoma cells (Pegues only very small colonies were fluorescent, and a and Stromberg, 1997). GSE screen hits were cloned into substantial fraction of those colonies have lost the a bicistronic tetracycline-inducible plasmid vector down- GFP fluorescence in the part of colony suggesting GSE stream from the hrGFP-IRES cassette. By measuring cytotoxicity. RNA levels, we have confirmed that expression of GFP To determine whether the observed functional effects correlates linearly with the putative GSE RNA expres- of inducible GSEs are due to downmodulation of their sion (data not shown). Therefore, we used GFP cognate genes, we analysed the IGF2R protein expres- fluorescence as expression marker for putative GSEs. sion by immunocytometry upon induction of GSE To isolate cell populations enriched for GFP and expression. Figure 3 shows that IGF2R GSE has a consequently putative GSE expressors, cells were treated slight downmodulation effect on IGF2R surface expres- for 16 h with doxycycline and FACS sorted to over 95% sion in HCT116 cells. We were unable to verify purity. downmodulation of SLC31A1 protein because no Interestingly, after 2 weeks of expansion of sorted commercial antibody against this protein is currently cells in the absence of doxycycline, only vector and available. Overall, the combined data obtained with putative IGF2R GSE cell lines retained GFP induci- inducible GSE cell lines corroborate the conclusion that bility by doxycycline (Figure 2a, 0-h time point), expression of L1CAM, SLC31A1 and IGF2R GSEs whereas the rest of the cell lines had lost GFP impairs tumor cell survival and/or proliferation. inducibility in significant portion of their populations (Figure 2a), suggesting that putative GSE expression is Cognate gene silencing by siRNA induce apoptosis cytotoxic. Upon further treatment with doxycycline for 2 more weeks, percentage of GFP-positive cells con- To confirm the doxycycline-inducible GSE phenotypes tinued to decrease in SLC31A1 putative GSE cell line, as by independent gene silencing approach, we have well as in c-ErbB2 antisense control line, whereas GFP designed siRNA inhibitors for L1CAM, SLC31A1 and expessing subpopulation remained relatively stable, IGF2R genes (Table 2). Target siRNAs were transiently albeit low, in L1CAM cell line. transfected into HCT116 cells and their effects on cell To evaluate the ability of individual putative GSEs to survival were analysed using caspase 3 and annexin V induce apoptosis in host cells, we have examined the assays. Transfection conditions were optimized to activation of caspase 3 in the putative GSE cell lines ensure at least 77% efficiency in HCT116 cells. after 48 h of doxycycline treatment (Figure 2b). Increase Apoptosis induction of fourfold or greater over control of 1.5-fold or greater in caspase 3 activation over vector (cells transfected with scrambled siRNA and exhibiting control was detected in GFP-positive subpopulations of 3% active caspase 3-positive cells) was detected for all the cell lines expressing putative L1CAM and SLC31A three siRNAs (Figure 4a, Table 3). The most potent GSEs. A small increase in caspase 3-positive cells was inducer of apoptosis was siRNA directed against seen in these cells even in absence of doxycycline. No L1CAM, a cell surface protein that plays an important significant apoptosis induction was observed in IGF2R role in axon guidance during nervous system develop- and c-ErbB2 antisense cells. ment, and which has been recently suggested to Next, we have examined short-term growth curves of contribute to the development and progression of the cell lines using Alamar blue assay (Figure 2c, d). As human malignant melanoma (Fogel et al., 2003). A role shown in Figure 2d, vector and IGF2R cell lines and for L1CAM in cancer is described in a separate

Oncogene Cell surface and secreted cancer targets MS Gelman et al 8163 manuscript (Suhy et al., submitted). Downmodulation activity was detected with siRNAs directed against of the L1CAM protein expression by respective siRNA IGF2R and SLC31A1. In case of IGF2R siRNA, a has been detected (Figure 4b). Comparable apoptogenic signiﬁcant inhibition of target protein expression was also observed (Figure 4b). Annexin V assay of HCT116 cells transfected with the same siRNAs has revealed apoptosis induction of about twofold over control (cells transfected with scrambled siRNA and exhibiting 11% annexin V-positive cells) for SLC31A1 siRNA (Figure 4a, Table 3). Surprisingly, transfection with siRNAs against L1CAM and IGF2R while causing detectable caspase 3 activation did not score in a more sensitive phosphatidylserine externaliza- tion annexin V assay. This effect could be due to variations in apoptotic response and/or kinetics induced by different siRNAs. Overall, all three target genes were conﬁrmed by siRNA inducing apoptosis detectable by at least one assay.

IGF2R GSE inhibits HCT116 tumor growth in xenograft mouse model We have further studied the effect of IGF2R GSE on tumor growth in HCT116 xenograft mouse tumor model. HCT116 cells expressing tetracycline-inducible IGF2R GSE were implanted in mice and allowed to form tumors. Upon tumor formation half of the animals were treated by doxycycline while the other half were kept untreated. Tumor growth was monitored for about 30 days under a continuous doxycycline treatment.

Figure 2 Survival and proliferation of stable HCT116 derivatives expressing tetracycline-inducible GSEs against select cell surface and secreted target genes. (a) Expression of GFP in the cell lines upon continuous induction with doxycycline (1 mg/ml) over a period of 2 weeks. Percentage of GFP-positive cells was measured by flow cytometry at days 1 (open bars), 7 (hatched bars) and 14 (solid bars) of the inducer treatment. Results of triplicate measurements are shown. (b) Induction of apoptosis in the cell lines treated with doxycycline (1 mg/ml) for 72 h as measured by active caspase 3 assay in GFP-positive cell subpopulations (solid bars). Untreated controls (total cell populations) are shown as open bars. Relative number of caspase 3-positive cells was determined by normalizing against untreated cells expressing the empty vector (exhibiting 1.6% of caspase-positive cells). Results of at least three independent experiments are presented. (c) Plating efficiency of the cell lines normalized to plating of the cells expressing empty vector. In total, 10 000 cells were plated in 24-well plate in triplicates, and plating efficiency was measured by Alamar blue assay 16 h after plating. (d) Relative proliferation rate of the cell lines grown in the presence of doxycycline (1 mg/ml). In all, 10 000 cells were plated in 24-well plate in triplicates, doxycycline was added to cells 16 h after plating, proliferation was measured by Alamar blue assay at 0 (open bars), 24 (hatched bars) and 48 (solid bars) hours post induction. Relative cell numbers was calculated for each cell line by normalizing against 0-h time point. (e) Relative soft agar colony number of the cell lines grown in the absence (open bars) or presence (solid bars) of doxycycline. A total of 5000 cells were plated in soft agar in the presence or absence of doxycycline (1 mg/ ml) and colonies were allowed to expand for 14–21 days prior to fixation and staining with crystal violet. Relative colony number was calculated for each cell line by normalizing against vector control. Results of three independent experiments are shown. (f) Flourescence view of soft agar colonies. Prior to crystal violet staining, soft agar colonies were examined by fluorescence microscopy. Representative visual fields are shown. The arrows mark colonies which lack GFP expression

Oncogene Cell surface and secreted cancer targets MS Gelman et al 8164 (Figure 5). In contrast, HCT116 cells expressing ErbB2 antisense construct showed inhibition of tumor growth independent of doxycycline induction. This is most likely due to a leakage in the transgene expression in the absence of doxycycline. These data indicate that IGF2R may play a role in tumor growth in vivo.

Discussion

Genes that play key roles in regulating cell growth and apoptosis could be used as molecular targets for cancer therapeutics. We developed a genomic screen to select target genes for induction of apoptosis in cancer cells. By making a fragmented transcriptome library, we were able to survey large number of genes and to select key genes that are implicated in apoptosis. In an attempt to achieve screen saturation we have analysed over 109 cells transduced with the library during the first iteration of the screen representing at least 20 library complexities. A significant number of multiple GSE screen hits per cognate gene were identified. This outcome suggests that the throughput of the screen was sufficient to cover substantial part of messages in the pool of the cancer cell transcriptomes. Target genes corresponding to multiple GSE screen hits encode a variety of proteins with diverse functions. Several genes known to be in the apoptosis pathways, DNA repair pathways and signal transduction molecules, for example, G-protein signaling components, have been captured by the screen. Interestingly, a large group of protein components of protein biogenesis machinery has been identified by the screen suggesting an indirect mode of action for such GSE screen hits resembling the effect of protein synthesis inhibitors. Our subsequent analyses of the hits were focused on a specific group of genes encoding surface and secreted proteins since they could be considered as targets for Figure 3 Effect of IGF2R GSE on the target protein expression. monoclonal antibodies and protein therapeutics. Con- HCT116 cell line with inducible expression of IGF2R GSE was comitant isolation of the three such genes, IGF2R, incubated in the absence or presence of doxycycline (1 mg/ml) for L1CAM and ITGA3, in the colon carcinoma apoptosis 48 h and stained with the corresponding APC-conjugated antibody. To verify the specificity of GSE-mediated protein downregulation, and breast carcinoma proliferation (Primiano et al., cells carrying empty vector expressing tetracycline-inducible 2003) GSE screens not only provides additional line of hrGFP were similarly processed and stained with the same evidence for validity of these cancer targets but also antibodies. (a) Anti-IGF2R-APC antibody fluorescence plotted raises important issue of pleiotropic effects of GSEs against hrGFP fluorescence. (b) IGF2R surface protein expression in total cell population. Isotype control antibody was used to against these targets on the both tumor cell proliferation determine nonspecific staining (solid gray lines, ‘isotype’). Solid and survival. dark lines correspond to untreated cells (‘Àdox’) and broken lines To further study and characterize the role of GSE correspond to doxycyline-treated cells (‘ þ dox’) screen hits in cell regulation, we generated stable cell lines expressing three selected putative GSEs. Analysis of apoptosis, plating efficiency, anchorage-independent Parent HCT116 cells were used to implant negative growth and proliferation as well as stability of GFP- control tumors and HCT116 cells expressing tetracy- IRES-GSE cassette expression (Figure 2) reveals a low cline-inducible ErbB2 antisense construct (Pegues and survival rate in all three cell lines; however, short-term Stromberg, 1997) were used as positive control. The growth curves show a clear cytostatic phenotype for median tumor growth curves suggested that doxycycline SLC31A1 GSEs (Figure 2d). Our results suggest that treatment resulted in the inhibition of tumor growth in mechanism of SLC31A1 GSE action is directly impli- HCT116 cells expressing IGF2R GSE as compared to cated in cell proliferation. Interestingly, in agreement doxycycline untreated control (data not shown) and with our data, it was previously suggested that cell cycle parental HCT116 cells expressing Tet-repressor inhibitors initially cause abnormal mitosis (mitotic

Oncogene Cell surface and secreted cancer targets MS Gelman et al 8165 Table 2 Coordinates of the tetracycline-inducible GSEs and sequences of the siRNA molecules Gene symbol Accession Tetracycline-inducible Sense strand siRNA sequence, 50–30 Antisense strand siRNA sequence, 50–30 siRNA 50 GSE coordinates, label 50–30

Scrambled NA NA CAGUCGCGUUUGCGACUGGdTdT CCAGUCGCAAACGCGACUGdTdT None Luciferasea M15077 NA CGUACGCGGAAUACUUCGAdTdT UCGAAGUAUUCCGCGUACGdTdT FITC IGF2R NM_000876 5179–4920 GUGCACCAGUGUUGAGAACdTdT GUUCUCAACACUGGUGCACdTdT Cy5 L1CAM NM_000425 1741–1529 GAAAGGUUCCAGGGUGACCdTdT GGUCACCCUGGAACCUUUCdTdT None SLC31A1 NM_001859 1389–1235 GGACUCAAGAUAGCCCGAGdTdT CUCGGGCUAUCUUGAGUCCdTdT None aPhotinus pyralis (firefly) luciferase (Elbashir et al., 2001a) catastrophe), and subsequently induce apoptosis (De- We have explored a gene with no established link to marcq et al., 1994). For instance, L1CAM GSEs isolated cancer, SLC31A1, using siRNA and inducible GSE in antiproliferation GSE screen in MDA-MB-231 cells approaches. Interference with SLC31A1 expression via were shown to induce abnormal mitosis (Primiano et al., GSE and siRNA affects growth of HCT116 colon 2003), while in our study L1CAM GSE induced carcinoma cells. Expression of SLC31A1 mRNA was apoptosis in HCT116 cells. The IGF2R GSE shows detected in a five out of five cancer cell lines tested. significant antitumor potency in vivo (Figure 5) as evident In summary, using GSE methodology, we identified a from the tumor xenograft study. Our data demonstrate number of novel genes that may represent useful targets the feasibility of using stable GSE cell lines in xenograft for antibody therapies. Our results suggest requirement tumor studies and validate the role of IGF2R in cancer. for the cell surface targets IGF2R, L1CAM and Our GSE library was constructed to bias towards SLC31A1 in tumor cell growth in vitro, and suggests that antisense cDNA fragments to minimize transdominant IGF2R is required for xenograft tumor growth in mouse peptides from sense cDNA fragments. Nevertheless, model. Our future research is aimed at functional analyses antisense GSEs can act not only on its cognate mRNA of different cancer cell lines, protein expression studies in but also through displacement of mRNA-binding normal/tumor tissue samples and, ultimately, develop- factors. Confirmation of GSE effects by siRNA inhibi- ment and testing of therapeutic antibody candidates. tion studies allows filtering out potential false positive GSE hits. While siRNA may also be false positive (Jackson et al., 2003), it is most likely that inhibition by Materials and methods both GSE and siRNA indicates a bona fide hit. In transient siRNA transfection studies, we have Cell lines confirmed three GSE target genes, inhibition of which leads to increased apoptosis in HCT116 cells as shown by A549, HCT116, MALME-3M, IGROV1, MCF7, AHCN, H460 cell lines were received from DCTD Tumor Repository caspase 3 marker and, in case of SLC31A1, by annexin V (National Cancer Institute, Frederick, MD, USA). FNX-Eco marker. HCT116 cells transfected with siRNAs show retrovirus packaging cell line was received from Dr Gary P apoptosis induction phenotypes similar to those of cell Nolan (Stanford University). HCT116–16 cells are derivative lines stably expressing inducible GSEs (Figures 2, 4, of HCT116 cell line engineered to express ecotropic retrovirus Table 3). Phenotype comparison of stable GSE cell lines receptor and LacI repressor as previously described (Chang and stable siRNA cell lines could be a useful future and Roninson, 1996). HT1080, SKOV3, MDA-MB-231 and study. PC3 cell lines were purchased from ATCC. IGF2R gene has been previously associated with cancer since it is often mutated or epigenetically Retroviral and plasmid vector construction modified in various tumors (Ouyang et al., 1997; Yamada et al., 1997; Kong et al., 2000; Jamieson et al., Vector V99, an IPTG-regulated retroviral expression vector, was derived from pLNXCO3 (Chang and Roninson, 1996) by 2003). Our data support findings of IGF2R GSEs in incorporating the following modifications. A cDNA encoding proliferation screen (Primiano et al., 2003) and IGF2R a truncated murine Lyt-2-a0 molecule (Nakauchi et al., 1985) antisense effect on survival of HSV1-infected cells (Zhou was used to replace the neomycin phosphotransferase gene in and Roizman, 2002). However, IGF2R role as a death pLNXCO3. The MCS of pLNXCO3 was modified by receptor for granzyme B during cytotoxic T-cell-induced subcloning a stuffer fragment that contained FseI, MluI and apoptosis (Motyka et al., 2000), apoptogenic effect of RsrII restriction sites, stop codons in all three reading frames IGF2R overexpression in colon carcinoma SW48 cells downstream of the RsrII site, and M13 sequencing and unique (Souza et al., 1999) and cytoprotective effect of IGF2R PCR primer sites flanking the new restriction sites. ribozyme in MCF7 breast carcinoma cells (Chen et al., Plasmid p1674, a plasmid vector used to engineer cell lines to 2002) appear to be contradictory to our results. A recent express tet-repressor, was constructed by replacement of the CMV promoter in pcDNA6-TR (Invitrogen, Carlsbad, CA, study of IGF2R overexpression (Schaffer et al., 2003) USA) with the EF-1a promoter (Invitrogen) by PCR. Plasmid reports opposing proliferation phenotypes in different p1704, a tet-regulated bicistronic expression vector, was prostate cancer cell lines offering potential explanation constructed through a series of PCR cloning steps in which a for such discrepancy. cassette of humanized Renilla hrGFP (Stratagene, La Jolla,

Oncogene Cell surface and secreted cancer targets MS Gelman et al 8166

Figure 4 Effect of siRNA inhibitors designed against a representative group of the cell surface and secreted target genes on survival and proliferation of HCT116 cells. (a) Induction of apoptosis in HCT116 cells 72 h after transient transfection with siRNAs. Percentage of apoptotic cells was assessed by active caspase 3 (open bars) and annexin V (filled bars) assays. Relative number of apoptotic cells was determined by normalizing against percent of apoptotic cells in scrambled siRNA transfectants. Results of 5–6 independent transfections are shown. (b) Downmodulation of target protein expression by siRNA. Panels show staining with antibodies against L1CAM in HCT116 cells and antibody against IGF2R in MDA-MB-231 cells. Staining profiles of the cells transfected with specific siRNA (thick line, ‘ þ siRNA’) and scrambled siRNA (thin line, ‘ÀsiRNA’) are shown. Staining with isotype control antibody is shown as dashed line (‘isotype’)

CA, USA), an ECMV IRES element (BD Clontech), and a TO (Invitrogen). The antisense c-erbB-2 cDNA in p1704 is 1.5 kb EcoRI antisense c-erbB-2 cDNA fragment (Pegues and ﬂanked by FseI and SﬁI sites to permit replacement of the c- Stromberg, 1997) derived from pHER436 (ATCC) was erbB-2 cDNA fragment by a putative GSE recovered from V99 assembled between the BamHI and NotI sites of pcDNA5/ using the primers p5x (50-TCT GCA GCC CAG GTA AAA

Oncogene Cell surface and secreted cancer targets MS Gelman et al 8167 Table 3 Functional analyses of selected group of putative GSE cognate genes Gene symbol Stable tetracycline-inducible GSE assays siRNA transient transfection assays

Functional activitya Target gene Functional activityc Target gene downmodulationb downmodulationd ABCDEFG

IGF2R ÀÀÀ+ + +(48) + À +(72)e L1CAM À +ndndndnd + À +(72) SLC31A1 + + + À nd nd + + nd aA – 50% or greater decrease in percent GFP-positive cells in course of 2-week doxycycline treatment (Figure 2a), B – 1.5-fold or greater increase in percent of caspase 3-positive cells as compared to vector control (in presence of doxycycline) (Figure 2b), C – 25% or greater decrease in cell proliferation of caspase 3-positive cells compared to vector control (Alamar blue assay, 48 h) (Figure 2d), D – twofold or greater decrease in soft agar colony formation as compared to vector control in presence of doxycycline (Figure 2e), E – inhibition of tumor growth in xenograft mouse model (Figure 5). bDecrease in protein level detected by immunocytometry, number of hours post doxycycline induction prior to the measurement is shown in parentheses (Figure 3). cF - 1.5-fold or greater increase in percent of caspase 3-positive cells as compared to cells transfected with scrambled siRNA (Figure 4a), G - 1.5-fold or greater increase in percent of annexin V-positive cells as compared to cells transfected with scrambled siRNA (Figure 4a). dDecrease in protein level detected by immunocytometry, number of hours post siRNA transfection prior to the measurement is shown in parentheses (Figure 4b). eIGF2R protein downmodulation by siRNA was detected in MDA-MB-231 cells

2500

2000

1500

1000 tumor weight, mg 500

0 01015202530355 doxycycline treatment, days

parent doxycycline IGF2R GSE doxycycline ErbB2 AS doxycycline

Figure 5 Effect of IGF2R GSE on growth of HCT116 tumor xenografts. HCT116 cell lines expressing the TetR protein only (open circles), or IGF2R-GSE (solid squares) and ErbB2-AS (open triangles) were implanted into athymic mice, upon tumor formation mice had doxycycline (1 mg/ml) added to their drinking water on the day 1 and tumors were allowed to grow for up to 35 days. Tumor growth inhibition curves are shown for groups of ﬁve animals

TTC GCT AGC CTC TAGT -30) and x7 (50-TTA CGA CGG USA). Equal amounts from each cell line were pooled and CCT GTC TGG CCT CAC TCA CTC ACG GAC CG-30). used for poly(A þ ) normalization as follows. Driver cDNA was synthesized by reverse transcription (Omniscript, Qiagen) of poly(A þ ) RNA captured from 100 mg of pooled total RNA Cell surface FACS analysis using Oligotex oligo-dTbeads (Qiagen), with the bead-bound Antibodies against extracellular domains of IGF2R (Santa oligo(dT) serving as primer. RNA was removed from the Cruz Biotech, Santa Cruz, CA, USA), XPR1 (Santa Cruz driver cDNA by heat denaturation and extensive washing. Biotech), L1CAM (clone UJ127, Labvision, Fremont, CA, Next, bead-bound driver cDNA was hybridized with 50 mgof USA) and COL4A2 (Chemicon, Temeluca, CA, USA) were pooled total RNA for 10 min at 371C. The supernatant above obtained from commercial sources. Downmodulation of the driver beads was collected, and normalized poly(A þ ) RNA IGF2R protein by siRNA in MDA-MB-231 cells was studied was extracted from the recovered supernatant using oligo-dT using FITC-conjugated anti-IGF2R antibody (clone MEM- beads. The extent of normalization was evaluated by 238, Serotec, Raleigh, NC, USA). Labeled cell populations semiquantitative RT–PCR analysis for three genes (b-actin, were analysed by FACS analysis (FACSCalibur, BD c-myc and E2F1) (Gudkov and Roninson, 1997) and was Bioscience, San Jose, CA, USA). judged sufﬁcient if the signals for each gene reached a maximum between 25 and 30 cycles. The normalized poly(A þ ) fractions were boiled for 3 min to randomly fragment the Random fragment cDNA library construction RNA, and RNA was reverse transcribed into cDNA (Gubler Total RNA was harvested from eight cell lines, A549, and Hoffman, 1983) using the SuperScript double-strand 0 HCT116, HT1080, MALME-3M, H460, MCF-7, IGROV1 cDNA synthesis kit (Invitrogen) and the primer AscN10 (5 - 0 and ACHN, using RNeasy Mini Kits (Qiagen, Valencia, CA, ACC CCT CAC TTG GCG CGC CN10-3 ). Double-strand

Oncogene Cell surface and secreted cancer targets MS Gelman et al 8168 random fragment cDNAs were ligated to linkers produced by was then turned off and allowed to cool to room temperature. annealing oligonucleotides L1 (50-GGC TGG GTG TGG HCT116 cells were plated the day before transfection with CGG ACC G-30) and L2 (50-CGG TCC GCC ACA CCC AG- antibiotic-free media into a six-well plate at a density of 5 Â 104 30). Random fragment inserts were amplified by 22 cycles of cells per well and each well was transfected with 5 mlof20mM PCR, using the primers P1 (50-ACC CCT CAC TTG GCG siRNA duplex mixture using 5 ml of Oligofectamine reagent CGC C-30) and P2 (50-GGC TGG GTG TGG CGG ACC G- (Invitrogen). Controls for the transfections included the 30), and the PCR products digested with AscI and RsrII Oligofectamine-mediated transfection of an equivalent quan- restriction endonucleases. Vector was prepared by digesting tity of a duplex with sequences directed against firefly V99 with MluI and RsrII restriction endonucleases, random luciferase or a nonspecific control duplex, a sequence that fragment cDNAs were ligated to 900 ng of digested vector, and was determined to be not present in mammalian systems by the ligation products were transformed into ElectroMax BLASTanalysis. An additional control included Oligofecta- DH10B bacterial cells (Invitrogen). A portion of the transfor- mine-mediated transfection of an equivalent volume of the mation was serially diluted and plated to estimate library annealing buffer. Transfection conditions were optimized to complexity, while the balance of the transformations were used ensure at least 77% efficiency in HCT116 cells. Specific to seed LB cultures grown at 301C for 16 h in a rotary shaker siRNA-mediated effects on targeted genes were confirmed by at 300 r.p.m. Library plasmid DNA was prepared using DNA a minimum of two independent experiments. Maxiprep kits (Qiagen). Insert diversity was evaluated by gel mobility analysis and sequencing of PCR products was Stable cell lines expressing inducible GSE amplified directly from the bacterial colony (Hofmann and Brian, 1991) using primers M13F17 (50-GTA AAA CGA CGG Stable cell lines with tetracycline-inducible expression of CCA GT-30) and p6x (50-TCT GCA GCC CAG GTA AAA individual GSEs were generated by transfection of the p1704 TTC GCT AGC CTC TAG T-30). The final transcriptome- vector carrying bicistronic hrGFP-IRES-GSE cassette into wide GSE plasmid library had a complexity of 4 Â 107 HCT116-TRex8 clonal cell line stably expressing tetracycline independent colonies or greater, with over 95% containing repressor (TetR) protein. TetR protein was expressed from an insert, with an average size of 0.4 kbp. pcDNA6/TR vector (T-RExt System, Invitrogen). At 48 h after transfection, cells were replated into selection medium containing 200 mg/ml hygromycin (Invitrogen) and 10 mg/ml Selected GSE library construction and analysis blasticidin S (Invitrogen). After 10–14 days of selection, 10–30 Putative GSEs were recovered from the integrated proviral selected colonies were pooled, expanded and hrGFP-IRES- DNA after each round of functional selection by PCR GSE expression was induced by doxycycline (BD Clontech) amplification using Thermozyme DNA polymerase (Invitro- treatment (1 mg/ml, 15 h). Induced cells were sorted (FACS- gen) and the Zea mays-derived primer pair p5 Â (50-TCT GCA Vantage, BD Bioscience) to isolate GFP-positive populations GCC CAG GTA AAA TTC GCT AGC CTC TAG T-30) and (SPs) at over 95% purity, which were further expanded in the p6x (50-GAG GAA CAA GGA AAG CTG AAT ACA CGA absence of doxycycline. Inducible expression of hrGFP–IRES- CCC GTG AT-30). Recovered PCR products from 0-, 24-, 48- GSE cassette was confirmed by FACS and real-time PCR and 72-h apoptotic cell harvesting time points were quanti- analysis. In the case of IGF2R GSE-expressing cells used for tated and equal amounts of PCR products from each time xenograft studies (see below), antibiotic-resistant cells were point were mixed. Pooled PCR products were digested with sorted by FACS for GFP expression in the absence of FseI and RsrII, subcloned into 150 ng V99 digested with FseI doxycycline induction: the resulting refined population (RP) and RsrII, and the resultant selected libraries characterized, was expanded and used for in vivo studies. propagated, and purified as described above. Equal number of recombinant clones corresponding to 0-, 24-, 48- and 72-h time Apoptosis and proliferation assays points were pooled to prepare select GSE library. For each select GSE library, over 95% of the colonies had inserts. Apoptosis induction in stable cell lines with inducible GSE Complexities of GSE libraries used in the second and third expression and in siRNA-transfected cells was measured by the rounds of screen were 5.0 Â 106 and 1.0 Â 106 independent active caspase 3 assay. Stable cell lines were plated in six-well clones or greater, respectively. The final selected GSE library dishes at 50 000 cells per well and GSE expression was induced constructed after three iterations of functional selection in by addition of 1 mg/ml doxycycline at 24 h after plating. HCT116-16 cells had a complexity of 1 Â 106 independent Following 72 h of doxycycline treatment cells were harvested, clones or greater, with an average insert size of 0.24 kbp. floating and attached cells were combined, fixed in Cytofix/ Sequencing was performed on DNA harvested from individual Cytoperm solution (BD Pharmingen) and stained with PE- colonies in this library, and the BLASTalgorithm was used to conjugated monoclonal rabbit antiactive caspase 3 antibody search the GenBank sequence database for similarities. (68655X, BD Pharmingen). Data were collected by FACS (FACSCalibur, BD Bioscience) and analysed using CellQuest (Beckton Dickinson) software. SiRNA-transfected cells were siRNA transfection divided in halves at 72 h post-transfection. One half was The siRNA complexes (Table 2) used in these studies were analysed by active caspase 3 assay as described above, another designed to according to the set of guidelines established by the half was analysed by annexin V staining cells using Guava- Tuschl laboratory (Elbashir et al., 2001b, c). Single strands of Nexint kit (Guava Technologies, Hayward, CA, USA). complementary 21-nucleotide RNA with an overhang of two Briefly, cells were resuspended in 50 ml of cold Nexin buffer deoxynucleotides on the 30 termini were synthesized (Genset, containing annexin V-PE and 7-AAD. After incubating for Evry, France) for each of the targets. Each strand was 20 min at 41C, samples were washed with cold Nexin buffer independently resuspended in annealing buffer (10 mM Tris- and resuspended in this buffer for analysis on the Guava PCA HCl, pH 8.3; 0.2 mM MgCl2;50mM KCl) at a final system (Guava Technologies). concentration of 20 mM. To generate annealed siRNA Effect of inducible GSE expression on proliferation of duplexes, equivalent volumes of each RNA strand were HCT116 cells was measured by Alamar blue assay. Briefly, combined and heated to 901C for 1 min in a heat block which cells were plated in 24-well dish at 10 000 cells per well in two

Oncogene Cell surface and secreted cancer targets MS Gelman et al 8169 sets of triplicates. After 16 h, one set of cells was grown in the dyes (Cy-Dye mono-functional NHS ester; Amersham, Piscat- absence of doxycycline treatment while another set of cells was away NJ, USA). Dye-conjugated cDNAs were purified from treated with 1 mg/ml doxycycline to induce GSE expression for free dye using the CyScribe GFX purification kit (Amersham), up to 48 h. At 0-, 24- and 48-h time points, cells were incubated and used as targets for hybridization to 70-mer oligonucleotide with Alamar Bluet reagent (Biosource International, Camar- microarrays (Agilent) custom manufactured to contain probes illo, CA, USA) for 4 h, and fluorescence emission was for 3531 different cell-surface and secreted genes. Arrays were measured at 595 nm on Analystt HT(Molecular Devices, hybridized for 16 h at 601C using an Agilent In Situ Sunnyvale, CA, USA). The 0-h time point data was used to Hybridization Kit as per the manufacturer’s instructions, determine the plating efficiency. washed 10 min in 6 Â SSC, 0.005% Triton X-102 at 221C, Soft agar assays were performed to evaluate the effect of 0.1 Â SSC, 0.005% Triton X-102 for 10 min at 41C, dried doxycycline-induced GSE expression on anchorage-indepen- under a stream of nitrogen, and scanned with an Agilent dent cancer cell growth. HCT-116-derived cell lines with Microarray Scanner. Hybridization signals were extracted with tetracycline-inducible expression of IGF2R, SLC31A1 GSEs Agilent Feature Extraction Software version 7.1 (Agilent) as well as ErbB2as control were resuspended at 5000 cells in which yielded the median of all pixel intensities for each 1.5 ml of warm media containing 0.35% BactoAgar (Difco feature. To facilitate comparison of gene expression levels Laboratories, Detroit, MI, USA), and doxycycline was added across cell lines, the intensity of the signal for a specific to final concentration of 1 mg/ml. Untreated control cells were oligonucleotide probe was normalized to the average intensity processed similarly but without the addition of doxycycline. of the signal from the two b-2-microglobulin probes for the Cells were plated on top of 3 ml of 0.5% agar in 6 cm dishes in given cell line. triplicates. Plates were incubated for 3 weeks in humidified chamber at 371C, 5% CO2. Cell colonies were first examined by fluorescence microscopy for GFP expression, then visua- Mouse xenograft studies lized by staining with 0.005% p-iodonitrotetrazolium violet in Mouse xenograft studies tested the in vivo tumor growth of 50% methanol for 1 h at room temperature, image-captured by parental HCT116 cell lines expressing the TetR protein only scanning and images were processed using Adobe Photoshop (control), or IGF2R-GSE and ErbB2-AS in athymic nude software (Adobe Systems, San Jose, CA, USA). mice in the presence or absence of doxycycline. Female athymic nude mice (Charles River Laboratories, Wilmington, RNA preparation and microarray hybridization MA, USA) were implanted subcutaneously with 107 HCT116

6 cloned tumor cells. At 6–7 days after implant, 14–17-week-old Total RNA was harvested from 4 Â 10 cells using a High Pure mice bearing established HCT116 cloned tumors were sorted RNA isolation kit (Roche Applied Science, Indianapolis, IN, into two groups of five mice each. For each study, Group 1 USA) according to the manufacturer’s instructions. A pool of mice were not treated and Group 2 mice received 1 mg/ml of 10 human cell line RNAs (Universal RNA; Stratagene, doxycycline in their drinking water. Tumors were measured Carlsbad, CA, USA) was used as a reference in all array using calipers twice a week, and tumor volumes were hybridizations. Quality control of the total RNA was calculated. Animals were euthanized when their tumors facilitated by the use of a microcapillary electrophoresis reached the predetermined 1500 mm3 end point tumor volume. system (Agilent 2100 Bioanlyzer, Agilent, Palo Alto, CA, Study durations were 33–48 days, and at the end of each study USA). For each array, amino-allyl labeled single-stranded all remaining animals were euthanized. cDNA was synthesized from 10 mg of total RNA or universal RNA using a commercially available kit (Agilent Fluorescent Direct Label Kit; Agilent Technologies, Palo Alto, CA, USA) Acknowledgements according to the manufacturer’s instructions, except that a We thank Patrick O’Connor, Serge Lichtsteiner, Paul Rejto, dNTP mix containing 5-[3-Aminoallyl]-20-deoxyuridine 50- Todd VanArsdale, Mike North, Stephen Bender and Stephen triphosphate (AA-dUTP; Sigma-Aldrich) was used (final Dunn for critical review and discussions of the data, Jerry concentration: 100 mM dATP, dCTP, dGTP; 50 mM dTTP, Barnhart for expert assistance with flow cytometry, Igor AA-dUTP). Amino-allyl labeled cDNA was purified using Roninson for a gift of pLNXCO3 vector, NCI for panel of QIAquick PCR columns (Qiagen, Valencia CA, USA) and tumor cell lines. Part of this work was carried out in coupled to either N-hydroxysuccinimidyl-esterified Cy3 or Cy5 collaboration with Pfizer, Inc. and Surromed, Inc.

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