High-throughput ectopic expression screen for tamoxifen resistance identifies an atypical kinase that blocks autophagy

Laura Gonzalez-Malervaa,b, Jaehong Parkb, Lihua Zouc, Yanhui Hub, Zahra Moradpoura,b,d, Joseph Pearlbergb, Jacqueline Sawyerb, Hallam Stevensb, Ed Harlowb,1, and Joshua LaBaera,b

aThe Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, AZ 85287; bHarvard Institute of Proteomics, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; cThe Dana Farber Cancer Institute, Boston, MA 02115; and dDepartment of Pharmaceutical Biotechnology, Faculty of Pharmacy, University of Medical Sciences, 71364 Shiraz, Iran

Contributed by Ed Harlow, December 13, 2010 (sent for review October 13, 2010)

Resistance to tamoxifen in breast cancer patients is a serious Kinases play an essential role in cellular physiology and several therapeutic problem and major efforts are underway to understand have been shown to cause tamoxifen resistance. It is likely that underlying mechanisms. Resistance can be either intrinsic or ac- other kinases contribute to hormone independence. quired. We derived a series of subcloned MCF7 cell lines that were either highly sensitive or naturally resistant to tamoxifen and studied Results the factors that lead to drug resistance. -expression studies Ectopic Kinase Expression Screen for Tamoxifen Resistance. revealed a signature of 67 that differentially respond to ta- Tamoxifen-sensitive and -resistant MCF7 subclones. We selected the moxifen in sensitive vs. resistant subclones, which also predicts MCF7 line for a cell-based screen because it requires estrogen for disease-free survival in tamoxifen-treated patients. High-throughput proliferation and is growth-inhibited by antihormone therapy. We cell-based screens, in which >500 human kinases were indepen- noted, however, that MCF7 displayed a heterogeneous response dently ectopically expressed, identified 31 kinases that conferred to hormonal manipulation, revealing partial but not complete cell drug resistance on sensitive cells. One of these, HSPB8, was also in killing after tamoxifen. Thus, we used limiting dilution to separate the expression signature and, by itself, predicted poor clinical out- seven well-behaved, subcloned cell lines of which three were sensitive and four were resistant to tamoxifen based on the ratio of come in one cohort of patients. Further studies revealed that HSPB8 A protected MCF7 cells from tamoxifen and blocked autophagy. More- growth in tamoxifen/control (Fig. 1 ). We used resazurin uptake (Fig. 1B) and microscopic observation (Fig. 1C) to further char- over, silencing HSBP8 induced autophagy and caused cell death. > Tamoxifen itself induced autophagy in sensitive cells but not in re- acterize two each of these that were either sensitive ( 90% cell death), MCF7-B7TamS and MCF7-C11TamS, or tamoxifen-re- sistant ones, and tamoxifen-resistant cells were sensitive to the in- sistant [proliferate in presence of 4-OHT (4-hydroxy-tamoxifen)], duction of autophagy by other drugs. These results may point to an MCF7-G11TamR and MCF7-H9TamR. The inhibitory concentra- important role for autophagy in the sensitivity to tamoxifen. tion for 50% of the sensitive cells (IC50) was 5 μM for tamoxifen (TAM) or 1 μM for 4-OHT after 6 d, whereas the resistant cells functional screen | estrogen receptor continued to grow at these conditions. We confirmed that the subclones were all derived from the he two thirds of women with estrogen receptor- (ER) or parental line by demonstrating 100% concordance with the Tprogesterone receptor-positive breast cancers are excellent parent and other MCF7 cells for a panel of 24 SNPs and less candidates for antihormone therapy. Selective ER modulators than 5% concordance across other non-MCF7 cells (Table S1). (SERMs), like tamoxifen, block ER activation and have impacted High-throughput functional cell-based screens. We screened our col- both therapy and survival. However, the success of tamoxifen lection of >500 full-length and fully-sequenced cDNAs of human therapy is limited by intrinsic and acquired drug resistance. Sev- kinases (14) to find those that confer tamoxifen resistance in the eral pathways have been implicated in antiestrogen resistance, sensitive subclones. We introduced the ectopic kinases using including: the PI3K/AKT/mTOR (mammalian target of rapamy- retroviral transduction because it was more efficient and showed A cin) pathway, which is implicated in cell survival; the EGFR less variability than transfection (Fig. S1 ). Assay conditions were family; and the RAS/RAF/MEK1/2/ERK1/2 family, which regu- adjusted using ERBB2 as a positive control because its ectopic overexpression conferred resistance to MCF7 (1) (Fig. S1B). late cell proliferation (1, 2). Loss of ER expression or function TamS may also be an important mechanism of de novo resistance to We adapted the MCF7-C11 for transduction in high- throughput screen (15). Using a partial set of 250 human kinases tamoxifen, either through relatively rare ER mutations or changes in a pilot screen, we established reproducible conditions on dif- in coactivators and corepressors (3). ferent days at different drug concentrations with a calculated Several groups have used gene-expression analysis to identify correlation coefficient of 0.80 (Fig. 1D). We then executed three genes regulated through ER (4) that are affected by SERMs in independent screens with the complete pJP1520-Kinase set breast cancer cells (5, 6). Others have used tumor samples to de- (505). From these and the pilot screen, we identified 80 kinases velop gene signatures that can predict clinical responses to tamox- that conferred resistance to 4-OHT (z score > 1.5). The viral – ifen (7 10). Genetic strategies have also been used to identify genes supernatants for these 80 chosen candidate kinases were prepared that drive tamoxifen resistance. Receptor tyrosine kinases and MAPK signaling were detected using expression of pooled cDNA

libraries in ZR-75-1, an approach often biased toward the most Author contributions: L.G.-M., J. Park, E.H., and J.L. designed research; L.G.-M., J. Park, abundantly expressed genes and which requires recovery of hits by L.Z., and Z.M. performed research; J. Pearlberg and J.S. contributed new reagents/analytic PCR (11). The analysis of antiestrogen-sensitive and -resistant tools; L.G.-M., L.Z., Y.H., H.S., and J.L. analyzed data; and L.G.-M. and J.L. wrote the paper. MCF7 cells by SNP and comparative genomic hybridization pointed The authors declare no conflict of interest. to changes in abundance rather than somatic genomic Freely available online through the PNAS open access option. changes (12). An RNA interference screen of kinases identified 1To whom correspondence should be addressed. E-mail: [email protected]. CDK10, CRK7, and MAP2K7, whose knockdown cause tamoxifen This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. resistance in MCF7 cells (13). 1073/pnas.1018157108/-/DCSupplemental.

2058–2063 | PNAS | February 1, 2011 | vol. 108 | no. 5 www.pnas.org/cgi/doi/10.1073/pnas.1018157108 Downloaded by guest on September 29, 2021 and resistant subclones by performing gene-expression profile analysis after estrogen and tamoxifen challenge. As expected in sensitive cells, tamoxifen blocked the ability of estrogen to induce (or repress) genes. We focused specifically on identifying the genes that failed to show an effect of tamoxifen in resistant cells. These 227 estrogen-responsive genes responded to tamoxifen in sensitive cells but not resistant ones (Fig. 2A). The genes included CCND1, IGF1R, MYC, and RERG, which are known to contribute to the development of resistance, as well as ERBB2, BCAR3, PIK3C2B, PIK3R3, and some negative regulators of cell cycle progression, such as CDKN1A (p21) and CDKN2B (p15) (1) (Table S4). We found that p53, TGFβ, p21 regulation, ErbB, cell cycle, and Jak-STAT signaling were the pathways that demonstrated statistically significant association with tamoxifen deregulation in resistant cells (Table S5). Clinical outcome prediction. We wondered if these 227 genes might also be predictive for outcomes in breast cancer patients who received tamoxifen. Because tumor tissue cannot be challenged with drugs, we focused on the subset of genes with an expression difference between the sensitive and resistant cells at baseline (no treatment). Using the PAM algorithm (17), we found 72 probes for 67 unique genes (Table S4). The entire microarray dataset is available through the Gene Expression Omnibus (ac- cession no. GSE 26459). Using clinical studies with long-term outcomes in tamoxifen- treated women that examined gene expression in tumors (18), the expression profile for each patient’s tumor was sorted into “re- sistance-like” or “sensitive-like” groups. These genes, selected entirely for their differential tamoxifen responses in our sensitive and resistant subclones, predicted better disease-free survival of patients that matched the “sensitive” signature (Fig. 2B). Pre- sumably, these patients were more likely to respond to tamoxifen and, hence, had a lower probability of relapse compared with patients whose patterns were closer to the resistant cells. In con- trast, the “resistance pattern” patients had a one in two chance of relapse after 5 y of tamoxifen treatment. We further confirmed C Fig. 1. MCF7 subclones and kinome screen. (A) MCF7 cells were diluted and this result using a different dataset (19) (Fig. 2 ). plated into 96-well plates. Condensed plates were split into three different Univariate analysis showed that the tamoxifen resistance sig- plates: archive, control, and tamoxifen. Cell proliferation was measured and nature was associated with a strong hazard ratio (2.12), compa- 12 subclones were selected. (B) Cell proliferation assay showed that resistant rable to tumor size (Table S6). Moreover, multivariate analysis subclones continued to proliferate in presence of 4-OHT (1 μM) and ta- demonstrated that as a prognostic predictor of relapse, the re- moxifen-sensitive cells were inhibited. (C) Photomicrographs of MCF7-B7TamS sistance signature was independent of the clinical variables and MCF7-G11TamR showed growth inhibition of sensitive but not resistant available in this study, including histological grade. cells. (D) Scatter plot of the z-scores showing the correlation of results for TamS kinase cDNA expression on cell growth of MCF7-C11 after 4-OHT at 1 Comparison of the Screen and Microarray Analyses for Pathways μ μ and 5 M and puromycin (1 g/mL). (E) Scatter plot of cell proliferation Involved in Tamoxifen Resistance. A number of genes were com- based on resazurin of 80 candidate kinases in the presence and absence of mon to both the cell based screen and the gene-expression analysis, 4-OHT (1 μM) and puromycin. including ERBB2, HSPB8, IGF1R, and SGK3. We focused our interest on HSPB8 because: it has never been previously studied in fi TamS TamS endocrine resistance; it repeated in ve of six screens; it gave one and tested on MCF7-C11 and MCF7-B7 six times in- of the strongest responses, and it was also present in the 67-gene dependently to narrow down the best candidate kinases. Growth signature that predicted relapse for women taking tamoxifen. in the presence of puromycin and in the absence of 4-OHT in- At baseline, HSPB8 was expressed at higher levels in the re- dicated good transduction efficiency (Fig. 1E). In the presence of sistant compared with the sensitive cells and it was not inhibited 4-OHT, the majority of these kinase genes enhanced cell pro- by tamoxifen in the resistant cells (Fig. 2D). Moreover, when the liferation by two- to threefold compared with the control-EGFP. 277 patients from Loi et al. (19) were sorted based on HSBP8 From the above criteria, 60 kinases among 80 were found to be expression alone, a high expression level of HSPB8 predicted an E positive in at least one experiment, and 31 were positive in at earlier relapse on tamoxifen (Fig. 2 ). A similar trend was ob- fi least four of six independent experiments (Table S2). A pathway served in the Miller dataset (18), but was not signi cant. analysis of the candidates that scored at least twice (47 in total) HSPB8 as an Important Protein in the Development of Tamoxifen

indicated 20 genes that enable tamoxifen escape (Table S3). MEDICAL SCIENCES Resistance. We established stable cell lines expressing 3xFlag- EGFR, ERBB2, and IGF1R are therapeutic targets for breast tagged versions of the protein in MCF7-B7TamS, with either car- cancer and inhibitors are already clinically available (16). We boxyl or amino-terminal modification, with the former showing fi also identi ed kinases, such as HSPB8, C9ORF96, and TRIB1 greater abundance (Fig. 3A). Compared with the luciferase con- that have relatively unknown functions and had not been asso- trol, ectopic expression of both HSPB8 constructs provided a ciated with tamoxifen resistance. proliferation advantage with 4-OHT treatment (Fig. 3B). Con- sidering its lower abundance, the amino-tagged construct was Microarray Expression Analyses of Tamoxifen-Sensitive and -Resistant more active than the carboxyl-tagged, perhaps because of the tag MCF7. We further compared the differences between the sensitive position. Neither was as active as the naturally resistant clones,

Gonzalez-Malerva et al. PNAS | February 1, 2011 | vol. 108 | no. 5 | 2059 Downloaded by guest on September 29, 2021 Fig. 3. Stable ectopic expression of HSPB8 blocks autophagy after tamoxi- fen. (A) Stable cell lines in MCF7-B7TamS expressing HSPB8 flag-tagged at either HSPB8C or HSPB8N were compared by immunoblot with MCF7-B7TamS and MCF7-G11TamR after 4-OHT (1 μM) for 72 h. Luciferase (Luc) was used as a negative control. (B) Cell proliferation assay of above cells was performed in 96-well plates. Data represent resazurin uptake in presence of 4-OHT di- vided by resazurin uptake in absence of drug. (*P < 0.07, **P < 0.05, ***P < 0.005 calculated based on Welch Two Samples t test.) (C) Western blot analysis of the phosphorylation status of MAP kinases. RAFP and ERK1/2P were elevated in HSPB8 and increased further in presence of 4-OHT. (D) Stable HSPB8 and MCF7-G11TamR cells were treated for 72 h with three MEK inhibitors: PD98059, MEK1/2, and U0126 and control (**P < 0.01, ***P < fi Fig. 2. Microarray expression pro les of tamoxifen-sensitive and -resistant 0.00001). (E) ERK1/2P and HSPB8 were measured by western-blot after MCF7- subclones. (A) Gene cluster analysis for E-regulated genes showed 115 in- G11TamR was treated with MEK inhibitors. duced and 112 repressed genes, which no longer respond to tamoxifen in resistant cells. Stimulated genes are shown in red, inhibited genes in green, with black intermediate between the two. (B) From the genes in A, a set of D fi fi (Fig. 3 ). However, U0126 signi cantly reduced cell proliferation 67 genes were selected to test prognostic signi cance. Kaplan-Meier survival of both cell lines, which may point to activities unrelated to MEK analysis from Miller’s dataset (18) showed 37 patients with early recurrence that contribute to this effect. Interestingly, the addition of U0126 compared with 29 showing better disease-free survival; and (C) 92 patients TamR showed better disease-free survival compared with 185 at high risk in Loi’s reduced HSPB8 protein levels in the MCF7-G11 cells, but dataset (19). (D) Expression of HSPB8 increased in both cell lines after E neither PD98059 nor MEK1/2 had this effect (Fig. 3E). addition, but is suppressed by tamoxifen in MCF7-B7TamS and not in MCF7- G11TamR.(E) A Kaplan-Meier curve for disease-free survival, sorted based HSPB8 Expression Is Essential for Cell Proliferation in MCF7 Cells. As only on HSPB8 expression, showed better outcome when expression is low. ectopic HSPB8 expression supported proliferation in tamoxifen- From this set, 67 of the 92 low-risk patients showed low expression of HSPB8 treated cells, we tested whether HSPB8 was required for pro- (73% overlap). Similarly, 114 of the 139 patients with high HSPB8 expression liferation in resistant cells. We observed that three out of four were in the high-risk group (82% overlap). targeted shRNAs reduced both HSPB8 protein (Fig. 4A) and cell proliferation (Fig. 4B) in MCF7-G11TamR. The same effect was observed in MCF7-H9TamR and T47D-G9TamR cells, although less pointing to other pathways that contribute to tamoxifen re- pronounced in the latter (Fig. 4C). After silencing HSPB8, cells sistance, consistent with our gene-expression studies. rounded up and lost cell-to-cell contact. To determine if HSPB8 were affecting known tamoxifen re- These data suggest that HSPB8 is essential to survival. Relatively sistance pathways, we evaluated MAP kinase signaling. Although P P little is known about HSPB8 signaling, which has been reported to p38 and JNK did not change with added tamoxifen or HSPB8 participate in both autophagy (21) and apoptosis (22). Silencing expression, RAFP and ERKP were induced by both, with a partic- fi P HSPB8 did not result in signi cant apoptosis, suggesting that this ularly strong combined effect for RAF . The effect on MEK was was not the predominant response causing cell death (Fig. S2A). mixed (Fig. 3C). To probe the contribution of the MEK pathway to However, using the MAP LC3 antibody, we observed a punctuate cell survival in tamoxifen, we used three different MEK inhibitors: pattern characteristic of ongoing autophagy with all of the hairpins PD98059, MEK1/2, and U0126 (20). At effective concentrations, (Fig. 4D). We confirmed autophagosome formation when we ex- neither PD98059 nor MEK1/2 had a marked effect on cell pro- amined these cells by transmission electron microscopy (EM), which liferation for either the naturally resistant cells or the HSPB8 cells revealed the characteristic double membrane structures (Fig. 4E).

2060 | www.pnas.org/cgi/doi/10.1073/pnas.1018157108 Gonzalez-Malerva et al. Downloaded by guest on September 29, 2021 Fig. 5. Effect of autophagy in sensitive and resistant cells. (A) Fluorescence Fig. 4. Reducing HSPB8 in MCF7-G11TamR leads to autophagy. (A) Protein images of quadruplicate experiments were taken after LC3 staining in MCF7- expression of HSPB8 was reduced with three of four different targeted B7TamS cells stably expressing either HSPB8 or luciferase (control). Total hairpins in MCF7-G11TamR compared with vector-expressing scrambled number of cells were counted in the presence and absence of 4-OHT (1 μM) sequences. (B) Silencing HSPB8 reduced cell proliferation of MCF7-G11TamR. after 72 h and expressed as a percentage of no treatment controls (**P < The effect was more pronounced in the presence of 4-OHT (1 μM). Resazurin 0.01). (B) HSPB8 reduced the percentage of autophagic cells (mean ± SD) data were normalized against scramble in the presence of puromycin and in (***P < 0.003). (C) Representative images from B showing reduction of absence of 4-OHT after 48 h postdrug treatment (**P < 0.001). (C) Hairpins autophagy. Blue: nucleus by Hoechst; red: actin by Phalloidin; green:

HSPB888 and HSPB895 reduced cell proliferation compared with scramble in autophagy marker anti-MAP LC3. (Scale bars, 50 μM.) (D) EM images showed two additional tamoxifen-resistant subclones, MCF7-H9TamR and T47D- the presence of clusters of autophagosomes after control cells were treated G9TamR, in the presence of 4-OHT (**P < 0.001). (D) Immunofluorescence with 4-OHT. Autophagosomes were markedly reduced when the cells were

images after silencing HSPB888 in all resistant subclones showed the char- ectopically expressing HSPB8. (Scale bar, 500 nm.) (E) Western blot analysis acteristic redistribution of anti-MAP LC3 into punctuate green structures showed lower expression levels of mTOR and HSPB8 in MCF7-G11TamR 72 h (B, F, and J, respectively), indicating active autophagy, but scrambled hairpin posttreatment with autophagy-inducing drugs: rapamycin (0.1 μM), showed only diffuse staining (A, E, and I). Blue: nucleus by Hoechst; red: actin LY294002 (5 μM), U0126 (10 μM), but not with 4-OHT (1 μM). (F) Short hairpin by Phalloidin; green: autophagy marker anti-MAP LC3. (Scale bars 50 μM.) RNAs directed at HSPB8, mTOR, or scramble were transduced into MCF7- (E) EM consistently identified a large number of autophagosomes contain- G11TamR cells. Protein expression levels of HSPB8, mTORP, p70S6kP, and 4EBP- ing organelles undergoing degenerative changes (arrows) in all of the cells 1P were measured 72 h postinfection. β-Actin served as a loading control. where HSPB8 was silenced (D, H, and L). (Scale bars, 500 nm.)

affects the mTOR pathway by inhibiting p70S6K, leading to In the converse experiment, we tested whether ectopically autophagy (23). This finding pointed to a potentially important expressed HSPB8 blocked autophagy. Consistent with our previ- role for autophagy, as well as apoptosis, in cell death triggered by ous results, HSPB8 conferred a twofold proliferation advantage tamoxifen. Indeed, we observed that tamoxifen induced both to tamoxifen-treated cells (Fig. 5A). In addition, HSPB8 led to processes in sensitive but not in resistant cells (Fig. S2B). Con- more than a threefold reduction in the number of cells displaying sistent with this finding, two other drugs that induce autophagy, autophagy (Fig. 5B), as indicated by MAP LC3 (Fig. 5C)andauto- LY294002 and rapamycin (24), also inhibited the growth of the MEDICAL SCIENCES phagosomes in EM (Fig. 5D). Thus, silencing HSPB8 increased resistant cells (Fig. S3) and reduced HSPB8 levels (Fig. 5E). autophagy and cell death, whereas ectopic expression reduced Many drugs that induce autophagy act through the mTOR autophagy and improved cell survival in the presence of tamoxifen. pathway. Interestingly, we observed that silencing HSPB8 did not affect the protein abundance of mTOR, nor did silencing mTOR HSPB8 and Autophagy. The role of autophagy in the effect of affect HSPB8 (Fig. 5F). Decreasing mTOR affected known down- HSPB8 suggested an interesting alternative explanation for why stream targets, including decreasing p70S6kP and the complex ef- U0126 had a more pronounced effect on the proliferation of re- fect of increasing the amount of 4EBP-1P, but shifting it to a faster sistant cells (Fig. 3D). Unlike the other MEK inhibitors, U0126 migrating band (25), confirming that the hairpins inhibited the

Gonzalez-Malerva et al. PNAS | February 1, 2011 | vol. 108 | no. 5 | 2061 Downloaded by guest on September 29, 2021 mTOR pathway compared with the control. Interestingly, silencing neither in any of the resistant subclones (Fig. S2B). The ectopic HSPB8 activated both p70S6kP and 4EBP-1P, suggesting that expression of HSPB8 in sensitive cells prevented the induction of HSPB8 may ordinarily inhibit these phosphorylations and pointing autophagy by tamoxifen (Fig. 5) and presumably conferred a pro- to related but different roles for HSPB8 and mTOR in autophagy. liferation advantage on the treated cells (Fig. 3B). A constant low level of autophagy has been suggested for cancer cells; therefore, Discussion it is possible that prevention of autophagy in cells may lead to SERMs such as tamoxifen are part of the standard treatment a general proliferation advantage that could even extend to un- regimen for many ER-positive breast cancer patients and have treated cells in culture. been demonstrated to reduce deaths. Unfortunately, many Autophagy is a catabolic process involving self-digestion of cel- patients become resistant by mechanisms that have been only lular organelles as a means of cell survival; however, if taken too far, partly characterized (1, 2). autophagy can lead to cell death (33, 34). The regulation of We used the estrogen-dependent and tamoxifen-responsive cell autophagy signaling is tightly linked to oncogenic signaling, with line MCF7 to explore tamoxifen resistance in a high-throughput both positive and negative roles in cancer. Like HSPB8, several screen that identified 31 kinases of 500 (6%) that conferred strong common oncogenes also inhibit autophagy, including: class I PI3K, tamoxifen resistance when ectopically expressed in at least four of AKT, mTOR,andBCL-2, arguing that oncogenes might prevent six experiments (Table S2). To generate a clean drug response for autophagy from limiting the ability of cancer cells to proliferate. a high-throughput screen, we reduced the previously reported Consistent with this notion, several tumor-suppressor genes, such heterogeneity in the MCF7 cells (6, 26) by limiting-dilution sub- as Beclin 1, DAPk, p53, PTEN,andTSC1/TSC2 stimulate autoph- cloning. A relatively large fraction of the cells (∼60%) grew out in agy (35). However, the well-described oncogenes Ras and MYC permissive conditions, of which about half were already resistant both stimulate autophagy, suggesting that this interplay is likely to to tamoxifen, suggesting that MCF7 cells do not require tamoxifen be complex and not completely understood (34, 36). conditioning because resistant cells are already present in the There is controversy about whether one should turn autophagy heterogeneous population (27, 28). This finding emphasizes the on or off to treat cancer. At least in the context here, when in- importance of caution when interpreting hormone-response hibition of autophagy promotes cancer-cell survival in response to experiments done on commonly available MCF7 cells. drugs, then induction of autophagic cell death could be predicted The availability of sister subclones enabled us to identify estrogen- to have therapeutic value (35, 37). responsive genes that were blunted by tamoxifen in a sensitive cell The suppression of either HSPB8 or mTOR induces autophagy, line, but not in a matched resistant one. Many of these genes and appears to do so independently, in as much as neither affects the (>90%) have not previously been linked to tamoxifen resistance, levels of the other. Silencing HSPB8 activated both 4EBP1and although about one-third of them could be traced in the literature to p70S6k, suggesting that HSPB8 may ordinarily inhibit these phos- SERMS. Some of these genes are not linked into any known phorylation events independently of mTOR (Fig. 5E). The crosstalk pathways or classifications (Table S5). This demon- between signaling pathways that activate or inactivate autophagy stration is unique in showing that the effect of tamoxifen on these remains under investigation (38). Recent publications suggest that genes is altered in resistant cells. Moreover, this response in cultured 4EBP-1 may have unrecognized actions that affect mTOR signaling cells was also relevant to clinical outcome in patients receiving or autophagy (39), and there are conflicting reports on the role of tamoxifen therapy. A 67-gene subset was used to predict tumor p70S6k to either activate or suppress autophagy, depending on the recurrence in two independent cohorts. The tamoxifen response cellular context (40). The strong effect that silencing HSPB8 has on signature was an independent predictor of outcome compared with downstream targets of mTOR that are known to participate in other clinical variables, and the hazard ratio was among the stron- autophagy is certainly intriguing. It will be interesting to determine gest observed for the study. Signatures like this one may help per- how these downstream effects are mediated and which ones are sonalize therapy by planning regimens of adjuvant therapy best important to the establishment of autophagy and cell death. The suited to each particular patient. expression of HSPB8 also affected RAF and ERK1/2 (Fig. 3C), and We found a 15% overlap of the genes when we compared our this might explain some degree of tamoxifen resistance. However, tamoxifen-resistance signature with those of Lippman et al. (7) and inhibition of the downstream ERK1/2 phosphorylation with drugs Chanrion et al. (8). Looking more broadly at our entire set of 227 (Fig. 3D) did not seem to negate the resistance to tamoxifen and the genes with an altered tamoxifen response, we identified 21 genes cells continued to proliferate despite almost complete inhibition of that overlapped across the tamoxifen resistance studies (Table S4). ERK1/2 at the higher drug dose, suggesting that there must be other A considerable body of evidence has emerged regarding the pathways acting here. ER, growth factor, and kinase-signaling pathways and their role in Clearly, the mechanism of action and specific interacting tamoxifen resistance (1). Thus, it was reassuring that many of the partners of HSPB8 still needs to be determined. 31 kinases identified here were in the MAPK\ERK1/2 and re- ceptor tyrosine kinases pathways (Table S3). Other might Materials and Methods contribute by affecting the cell cycle (ATR, AURKA, CDC2, MCF7 Subclones. MCF7 cells were diluted into 10 96-multiwell plates (1/3 cell/ CDK9, and STK6) or metabolism (AK5, AK2, NME3, NME4, well) and grown for approximately 1 mo, including several changes of DMEM NME7, KHK, RBKS, PMVK). 10% FBS without selection. For those wells in which cells grew to confluence HSPB8 is a 22-kDa member of the small heat-shock protein (∼192), cells were trypsinized and condensed into two plates. After condensing superfamily, which contains a well-conserved α-crystallin domain all subclones, they were grown again to confluence and split into three dif- at the C terminal (29). HSPB8 has not been described in con- ferent plates: archive, control, and tamoxifen. After 6 d posttreatment, cell nection with tamoxifen resistance, but it is overexpressed in breast proliferation was measured by MTS Assay (Promega). We selected 12 subclones cancer, particularly in ER-positive cancers (6, 30). for further analysis, and from those, seven that behaved well in culture. Ta- The ectopic expression of HSPB8 enabled cell proliferation in moxifen-sensitive cells were regularly maintained in DMEM supplemented with 5% of FBS, and resistant cells were grown in same media supplemented the presence of tamoxifen in sensitive cells, although not to the with 4-OHT (1 μM). level of naturally resistant MCF7 cells. In addition to HSPB8, our gene-expression studies pointed to numerous genes that were also High-Throughput Functional Cell-Based Screens. Kinase plasmids were fully aberrantly regulated by tamoxifen in these resistant subclones, sequence verified and validated (14). DNA preparation, transfections, and many of which are known to play key roles in the oncogenic virus preparation have been described previously (15). For data analysis, we phenotype, including cyclin D1, myc, myb, p21, ERBB2, and p15, always subtract the signal obtained from an empty well and considered this which might contribute to resistance. as the background reading. Any wells whose cell viability in the presence of Interestingly, silencing HSPB8 in tamoxifen-resistant cells led to puromycin was less than 30% of those in the absence of puromycin were cell death via autophagy, not apoptosis (Fig. 4D and Fig. S2A). considered to have an inadequate viral titer, called dropouts (Fig. S1D). To Tamoxifen induces both processes in sensitive cells (31, 32) but identify genes that provided a growth advantage in the presence of ta-

2062 | www.pnas.org/cgi/doi/10.1073/pnas.1018157108 Gonzalez-Malerva et al. Downloaded by guest on September 29, 2021 moxifen, a z-score was computed for each well [(well fluorescence − mean of instruments, John Doench and Dorre Gruenberg for manuscript suggestions all well fluorescence for the plate)/SD of the mean of well fluorescence]. The and corrections, Dorre Gruenberg for providing the shRNA construct for genes for which the z score against entire plate was higher than z total > 1.5 mammalian target of rapamycin, and Milen Vitanov for his technical support were considered for further analysis. at Arizona State University. This work was supported by the National Cancer Complete details of methods are available in SI Materials and Methods. Institute Specialized Programs of Research Excellence in Breast Cancer at Harvard, the Breast Cancer Research Foundation, and the National Cancer ACKNOWLEDGMENTS. We thank Joan Brugge for providing MCF7 cells, Institute Program Project Grant P01 C080111. The Expedition Inspiration for Mauricio Fernandez for all the engineering technical support with the robot Breast Cancer Research supported L.G.-M.

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