LMTK3 Is Implicated in Endocrine Resistance Via Multiple Signaling Pathways

ORIGINAL ARTICLE LMTK3 is implicated in endocrine resistance via multiple signaling pathways

J Stebbing1, A Filipovic1, LC Lit1, K Blighe2, A Grothey1,YXu1, Y Miki3, LW Chow3,4, RC Coombes1, H Sasano3, JA Shaw2 and G Giamas1

Resistance to endocrine therapy in breast cancer is common. With the aim of discovering new molecular targets for breast cancer therapy, we have recently identified LMTK3 as a regulator of the estrogen receptor-alpha (ERa) and wished to understand its role in endocrine resistance. We find that inhibition of LMTK3 in a xenograft tamoxifen (Tam)-resistant (BT474) breast cancer mouse model results in re-sensitization to Tam as demonstrated by a reduction in tumor volume. A whole genome microarray analysis, using a BT474 cell line, reveals genes significantly modulated (positively or negatively) after LMTK3 silencing, including some that are known to be implicated in Tam resistance, notably c-MYC, HSPB8 and SIAH2. We show that LMTK3 is able to increase the levels of HSPB8 at a transcriptional and translational level thereby protecting MCF7 cells from Tam-induced cell death, by reducing autophagy. Finally, high LMTK3 levels at baseline in tumors are predictive for endocrine resistance; therapy does not lead to alteration in levels, whereas in patient’s plasma samples, acquired LMTK3 gene amplification (copy number variation) was associated with relapse while receiving Tam. In aggregate, these data support a role for LMTK3 in both innate (intrinsic) and acquired (adaptive) endocrine resistance in breast cancer.

Oncogene (2013) 32, 3371–3380; doi:10.1038/onc.2012.343; published online 6 August 2012 Keywords: LMTK3; breast cancer; estrogen receptor; endocrine resistance; HSPB8; oncogene

INTRODUCTION previously described to have a role in ERa-positive breast cancer. With an annual incidence of 1.3 million cases worldwide and We demonstrate that inhibition of LMTK3 decreased the total 465,000 deaths, breast cancer remains the most frequently protein levels of HSBP8 (HSP22), a member of the heat-shock diagnosed type of cancer and the leading cause of cancer protein family, recently identified as a potential target for Tam 11 mortality in females.1 Because ERa is expressed in over 70% of resistance via a high-throughput ectopic expression screen. breast cancers, it is not surprising that many treatments thus far Overexpression of LMTK3 promoted cell survival in MCF7 cells have focused on targeting ERa, directly or indirectly, by inhibiting (Tam sensitive) after Tam treatment by reducing autophagy, its activity, stability and/or biosynthesis.2–5 Nevertheless, many suggesting a potential mechanism by which LMTK3 acts in the patients become resistant to these treatments and relapse at a development on Tam resistance. rate of 1–1.5% of those diagnosed per annum. Although Resistance is typically innate (intrinsic) or acquired (adaptive), mutations in the ERa are rarely found,6 other mechanisms both factors that limit the success of therapies in the clinic. have been associated with tamoxifen (Tam) resistance, namely Whereas the latter is more common, we have previously shown in phosphorylation of ERa.7 Thus, deciphering the role of kinases in a large cohort that LMTK3 levels are predictive, as well as modulating ERa activity may derive new druggable targets with prognostic, in terms of response to Tam. Now, we have compared the potential to reverse this resistance, including re-sensitizing LMTK3 levels in responding breast cancers treated with endocrine cells to Tam.8,9 Using a high throughput screen with activity of therapy versus non-responding breast cancers during neo- 12,13 estrogen-responsive genes as a quantitative read-out, we have adjuvant treatment. Higher LMTK3 levels at baseline were recently identified Lemur Tyrosine Kinase 3 (LMTK3), for which a associated with tumors that were less likely to respond to therapy. role had not previously been assigned, as a ‘master’ oncogenic We have recently used SNP6.0 arrays to profile the ‘circulating regulator of ERa with transcriptional and translational effects.10 cancer genome’ comparing circulating free DNA (cfDNA) samples To establish a role of LMTK3 in reversing endocrine resistance, isolated from plasma with the primary tumor in 50 breast cancer we examined the effects of its inhibition in a Tam-resistant patients during follow-up. Specific copy number variations (CNVs) xenograft model and subsequent sensitivity to Tam. To under- were detected in cfDNA, mirroring the primary tumor, up to stand the signaling pathways implicated, we performed a twelve years after diagnosis despite no other evidence of disease, 14 genome-wide gene expression analysis using the Tam-resistant suggesting dormancy in the majority of patients. Because of the cell line (BT474) with small interfering RNA (siRNA) knockdown of high density of probes, 906 600 for SNPs and 946 000 for CNVs, it is LMTK3, which revealed that LMTK3 is able to regulate the possible to interrogate SNP/CNV data for most genomic intervals. transcription of several genes, some of which have been We therefore assayed CNV data in the LMTK3 gene in these

1Department of Surgery and Cancer, Division of Cancer, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London, UK; 2Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester, UK; 3Department of Pathology, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan and 4UNIMED Medical Institute, Comprehensive Centre for Breast Diseases, Hong Kong. Correspondence: Dr G Giamas, Department of Surgery and Cancer, Hammersmith Hospital Campus, Imperial College London, Du Cane Road, London W12 0NN, UK. Email: [email protected] Received 4 June 2012; revised 20 June 2012; accepted 21 June 2012; published online 6 August 2012 LMTK3 is implicated in endocrine resistance J Stebbing et al 3372 patients and observed LMTK3 gene ampliﬁcation in both cfDNA and primary tumor. Together, these data implicate oncogenic activation of LMTK3 in a ‘dual hit’ model, promoting both tumor growth and as a route to intrinsic endocrine resistance.

RESULTS Inhibition of LMTK3 re-sensitizes endocrine-resistant cells to Tam in vivo Having already shown that knockdown of LMTK3 restores the Tam growth inhibitory effects in various Tam-resistant breast cancer cell lines,10 we wished to investigate the involvement of LMTK3 in the development of endocrine resistance in vivo. Therefore, we used the BT474 cell line,15 which expresses high endogenous LMTK3 messenger RNA (mRNA) and protein levels compared with other cell lines (Supplementary Figure 1), to generate orthotopic breast cancer xenografts models. As expected, Tam-treated mice showed no changes in tumor volume compared with controls (P ¼ 0.63). Similar results were obtained when comparing control versus siLMTK3-treated mice (P ¼ 0.59). However, in the presence of both siLMTK3 and Tam, a signiﬁcant inhibition of tumor growth was demonstrated compared with untreated controls (P ¼ 0.03), suggesting that inhibition of LMTK3 re-sensitizes cells to Tam in vivo (Figure 1), a numerically modest but statistically signiﬁcant effect.

Inhibition of LMTK3 activates cellular pathways of endocrine responsiveness To identify signaling pathways relevant to endocrine responsiveness that LMTK3 may be implicated in, we performed a microarray-based genome-wide gene expression analysis using BT474 cells transfected with siControl or siLMTK3 in the presence or absence of different treatments (ETOH, E2 and E2 þ Tam), to simulate the treatment conditions used in the xenograft mouse model, recapitulating the physiological presence of E2 in female mice. Initially, we identified genes that were significantly altered between any two groups defined by siRNA transfection (siCTRL, Figure 1. Orthotopic breast cancer xenograft model. (a) BT474 cells siLMTK3) and treatment (ETOH, E2, E2 þ Tam) by ANOVA analysis were injected subcutaneously into the mammary fat pad of nude mice, grouped (n ¼ 10/group) according to treatment (siControl, (BH-Q false discovery rate (FDR)o0.05). Principal component siLMTK3, siControl þ Tam, siLMTK3 þ Tam), and per cent difference analyses using all genes from the ANOVA analysis showed that in tumor volumes were measured at the indicated time points. the strongest effects on expression level were induced by E2 (P ¼ 0.03: siControl vs siLMTK3 þ Tam, P ¼ 0.59: siControl vs siLMTK3, treatment (Supplementary Figure 2). We then constructed a heat P ¼ 0.63: siControl vs siControl þ Tam). (b) Histological analyses of map ranking genes according to degree of expression change LMTK3 and Ki67 expression in representative tumor tissue sections upon E2 treatment. Figure 2a and Table 1 show the top 83 genes from different groups. Original magnification, x200. Scale bar, induced or repressed by E2 treatment. We found well-known E2 100 mm. responsive genes such as PGR, GREB1, and TFF1 among the genes strongly induced by E2 in BT474 cells demonstrating that BT474 cells have retained functional ERa signaling. The observed E2 predictive of poor outcome following Tam therapy;20,21 HEY2,a response was reversed to some degree by Tam treatment. direct target gene of the Notch signaling pathway, the activity of However, for a limited number of genes (for example, OLFM1, which is enhanced in endocrine resistant breast cancers;22,23 MYBL1, GPR68, PDLIM3), a near-complete reversal of E2-induced SIAH2, expression of which predicts disease-free survival in Tam- expression change was observed (Supplementary Excel file 1). In treated patients,24 HSPB811), as well as genes that have not thus contrast, depletion of LMTK3 had much stronger effects resulting far been described as associated with breast cancer (for example, in an almost completely abrogated E2 response over a wide range GPR68, a member of the family of ovarian cancer G-protein- of genes (Figure 2b and Supplementary Excel file 2). coupled receptors that stimulate a variety of intracellular signaling Among those genes, there were several that have been pathways;25,26 and RASGRP1 that belong to the Ras guanyl previously reported to be implicated in breast cancer progression, nucleotide releasing proteins that may contribute to notably EGLN2 that regulates cyclin D1 and cell cycle progression hematologic malignancies27). in a hypoxia-inducible factor-dependent manner,16 LAPTM4B that Next, we searched for genes that are regulated by combined has a role in the development of de novo resistance to treatment with Tam and LMTK3 depletion, compared with siCTRL- anthracycline-based chemotherapy,17 RARA that is co-amplified treated cells, revealing various significantly downregulated and in a subset of ERBB2 þ breast cancers,18 UGT2B15 that reduces upregulated genes (Figure 2c and Supplementary Excel file 3). estrogen-mediated signaling in breast cancer cells,19 and also Finally, to identify genes that are relevant for re-sensitizing cells to those known to be relevant in the development of endocrine Tam, we compared genes differentially regulated by the synergistic resistance (for example, c-myc, high protein levels of which are treatment of Tam and LMTK3 depletion to the effects of either of

Oncogene (2013) 3371 – 3380 & 2013 Macmillan Publishers Limited LMTK3 is implicated in endocrine resistance J Stebbing et al 3373

Figure 2. Microarray analysis and comparison of genes regulated by LMTK3 under different treatments. (a) Heat map of genes that were significantly regulated ( þ or À ) after E2 treatment in the presence of siControl or siLMTK3 (Po0.05). (b) Effects of LMTK3 depletion on E2-regulated genes (measured as fold change in siCTRL-E2 vs siLMTK3-E2-treated BT474 cells). (c) Fold changes of genes after examining the combined effects of Tam treatment under LMTK3 depletion (measured as fold change in siCTRL-E2 vs siLMTK3-E2 þ Tam-treated BT474 cells). (d) Fold changes of genes after examining the effects of Tam treatment in LMTK3-depleted cells (measured as fold change in siLMTK3-E2 vs siLMTK3-E2 þ Tam-treated BT474 cells). these treatments alone (Figure 2d and Supplementary Excel file 4). siControl-E2/Tam, respectively (Table 2 and Supplementary Excel file Out of the 25 genes mostly affected upon Tam treatment in LMTK3- 4). Among those genes were some that have been implicated in depleted cells (candidates for synergism), a subset of 11 genes various types of malignancies, including (i) MPPED2, previously emerged, for which expression changes between siControl-E2 and linked to tumorigenesis in neuroblastoma;28 (ii) FAM3B, which is siLMTK3/E2/Tam were larger compared with the sum of effects relevant for pancreatic neuroendocrine tumors and is linked to between siControl-E2 versus LMTK3/E2 and siControl-E2 versus glucose regulation;29 (iii) FGD3, a guanine nucleotide exchange factor

& 2013 Macmillan Publishers Limited Oncogene (2013) 3371 – 3380 LMTK3 is implicated in endocrine resistance J Stebbing et al 3374 Table 1. List and references of top 83 genes that were induced or repressed after E2 treatment

Gene symbol Description Uniport KB RefSeq

ABCC3 ATP-binding cassette, sub-family C (CFTR/MRP), member 3 P78543 NP_006754.1. ADD3 Adducin 3 (gamma) Q9UEY8 NP_001112.2. AGPAT9 1-acylglycerol-3-phosphate O-acyltransferase 9 Q53EU6 NP_001243350.1. AGR3 Anterior gradient homolog 3 Q8TD06 NP_789783.1. ATXN1 Ataxin 1 P54253 NP_000323.2. BAMBI BMP and activin membrane-bound inhibitor homolog (Xenopus laevis) Q13145 NP_036474.1. BCAS1 Breast carcinoma ampliﬁed sequence 1 Q9HCH5 NP_001156423.1. BTG2 BTG family, member 2 P78543 NP_006754.1. CALCR Calcitonin receptor P30988 NP_001158209.1. CBLN2 Cerebellin 2 precursor Q8IUK8 NP_872317.1. CCNG2 Cyclin G2 Q16589 NP_004345.1. CD44 CD44 molecule P16070 NP_000601.3. CDH2 Cadherin 2, type 1, N-Cadherin (neuronal) P19022 NP_001783.2. CGNL1 Cingulin-like 1 Q0VF96 NP_001239264.1. COL12A1 Collagen, type XII, alpha 1 Q99715 NP_004361.3. CX3CL1 Chemokine (C-X3-C motif) ligand 1 P78423 NP_002987.1. CXCL12 Chemokine (C-X-C motif) ligand 12 P48061 NP_000600.1. CXCR7 Chemokine (C-X-C motif) receptor 7 P25106 NP_064707.1. DEGS1 Delta(4)-desaturase, sphingolipid 1 O15121 NP_003667.1. DEPDC6 DEP domain containing MTOR-interacting protein Q8TB45 NP_073620.2. EGLN2 Egl nine homolog 2 Q96KS0 NP_444274.1. FGD3 FYVE, RhoGEF and PH domain containing 3 Q5JSP0 NP_001077005.1. GPR68 G protein-coupled receptor 68 Q15743 NP_001171147.1. GREB1 TGrowth regulation by estrogen in breast cancer 1 Q4ZG55 NP_055483.2. HEY2 Hairy/enhancer-of-split related with YRPW motif 2 Q9UBP5 NP_036391.1. HMGCS2 3-Hydroxy-3-methylglutaryl-CoA synthase 2 (mitochondrial) P54868 NP_001159579.1. HOPX HOP homeobox Q9BPY8 NP_001138931.1. HSPB8 Heat shock 22kDa protein 8 Q9UJY1 NP_055180.1. IGFBP3 Insulin-like growth factor binding protein 3 Q96NL0 NP_001127877.1. IGFBP4 Insulin-like growth factor binding protein 4 P22692 NP_001543.2. ILMN_1874323 KCNJ13 Potassium inwardly-rectifying channel, subfamily J, member 13 O60928 NP_001165887.1. KIAA1199 KIAA1199 Q8WUJ3 NP_061159.1. KLK12 Kallikrein-related peptidase 12 Q86SQ0 NP_001127909.1. LAPTM4B Lysosomal protein transmembrane 4 beta Q86VI4 NP_060877.3. LOC730517 LOXL4 Lysyl oxidase-like 4 Q96JB6 NP_115587.6. LRRN1 Leucine rich repeat neuronal 1 Q6UXK5 NP_065924.3. MAN1A1 Mannosidase, alpha, class 1A, member 1 P33908 NP_005898.2. MME Membrane metallo-endopeptidase P08473 NP_000893.2. MPPED2 Metallophosphoesterase domain containing 2 Q15777 NP_001575.1. MYBL1 v-myb myeloblastosis viral oncogene homolog (avian)-like 1 P10243 NP_001073885.1. MYC v-myc myelocytomatosis viral oncogene homolog (avian) P01106 NP_002458.2. NDRG1 N-myc downstream regulated 1 Q92597 NP_001128714.1. NDUFB4 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 4 O95168 NP_001161803.1. NLGN4X Neuroligin 4, X-linked Q8N0W4 NP_065793.1. NXPH1 Neurexophilin 1 P58417 NP_689958.1. OLFM1 Olfactomedin 1 Q99784 NP_006325.1. PDLIM3 PDZ and LIM domain 3 Q53GG5 NP_001107579.1. PGR Progesterone receptor P06401 NP_000917.3. PHLDB2 Pleckstrin homology-like domain, family B, member 2 O75363 NP_003648.2. PMAIP1 Phorbol-12-myristate-13-acetate-induced protein 1 Q13794 NP_066950.1. PPFIBP2 PTPRF interacting protein, binding protein 2 (liprin beta 2) Q8ND30 NP_003612.2. PSCA Prostate stem cell antigen O43653 RAP1GAP RAP1 GTPase activating protein P47736 NP_001139129.1. RAPGEFL1 Rap guanine nucleotide exchange factor (GEF)-like 1 Q9UHV5 NP_057423.1. RARA Retinoic acid receptor, alpha P10276 NP_000955.1. RASGRP1 RAS guanyl releasing protein 1 (calcium and DAG-regulated) O95267 NP_001122074.1. RBP1 Retinol binding protein 1, cellular P09455 NP_002890.2. REG4 Regenerating islet-derived family, member 4 Q9BYZ8 NP_001152824.1. RUNDC3B RUN domain containing 3B O15438 NP_001137542.1. SCGN Secretagogin, EF-hand calcium binding protein O76038 NP_008929.2. SERPINI1 Serpin peptidase inhibitor, clade I (neuroserpin), member 1 Q99574 NP_001116224.1. SIAH2 Seven in absentia homolog 2 O43255 NP_005058.3. SLC7A5 Solute carrier family 7 (amino acid transporter light chain, L system), member Q01650 NP_003477.4. STARD13 StAR-related lipid transfer (START) domain containing 13 Q9UKR0 NP_062544.1. STC2 Stanniocalcin 2 O76061 NP_003705.1. SUSD3 Sushi domain containing 3 Q96L08 NP_659443.1. SYNPO2L Synaptopodin 2-like Q9H987 NP_001107605.1. SYT1 Synaptotagmin I P21579 NP_001129277.1. SYTL2 Synaptotagmin-like 2 P17936 NP_000589.2.

Oncogene (2013) 3371 – 3380 & 2013 Macmillan Publishers Limited LMTK3 is implicated in endocrine resistance J Stebbing et al 3375 Table 1 (Continued )

Gene symbol Description Uniport KB RefSeq

TBX2 T-box 2 Q13207 NP_005985.3. TFF1 Trefoil factor 1 P04155 NP_003216.1. TGFBR2 Transforming growth factor, beta receptor II P37173 NP_001020018.1. TMEM158 Transmembrane protein 158 (gene/pseudogene) Q8WZ71 NP_056259.2. TP53INP1 Tumor protein p53 inducible nuclear protein 1 Q96A56 NP_001129205.1. TP53INP2 Tumor protein p53 inducible nuclear protein 2 Q8IXH6 NP_067025.1. TPD52L1 Tumor protein D52-like 1 Q16890 NP_001003395.1. TRIB1 Tribbles homolog 1 (Drosophila) Q96RU8 NP_079471.1. UGT2B15 UDP glucuronosyltransferase 2 family, polypeptide B15 P54855 NP_001067.2. VASN Vasorin Q6EMK4 NP_612449.2. VTCN1 V-set domain containing T cell activation inhibitor 1 Q7Z7D3 NP_001240779.1. ZNF467 Zinc ﬁnger protein 467 Q7Z7K2 NP_997219.1.

Table 2. List and references of 11 candidate synergistic genes

Gene symbol Description Uniport KB RefSeq

C6orf141 Chromosome 6 open reading frame 141 Q5SZD1 NP_001139124.1. FAM3B Family with sequence similarity 3, member B P58499 NP_478066.3. FGD3 FYVE, RhoGEF and PH domain containing 3 Q5JSP0 NP_001077005.1. ID1 Inhibitor of DNA binding 1, dominant negative helix-loop-helix protein P41134 NP_002156.2. ID4 Inhibitor of DNA binding 4, dominant negative helix-loop-helix protein P47928 NP_001537.1. INPP4B Inositol polyphosphate-4-phosphatase, type II O15327 NP_001095139.1. LOC645726 MPPED2 Metallophosphoesterase domain containing 2 Q15777 NP_001575.1. SGK3 Serum/glucocorticoid regulated kinase family, member 3 Q96BR1 NP_001028750.1. TCN1 Transcobalamin I (vitamin B12 binding protein, R binder family) P20061 NP_001053.2. TSKU Tsukushi small leucine rich proteoglycan homolog (Xenopus laevis) Q8WUA8 NP_056331.2. for Cdc42, involved in cell migration;30 (iv) ID1, expression of which is expression of HSPB8. As previously described, E2 or Tam associated with more invasive features of cancer and with the treatment increased HSPB8 protein levels;37 however, this epithelial-mesenchymal transition;31 (v) ID4, which is associated increase was abrogated after inhibition of LMTK3 (Figure 4a). with shorter recurrence-free survival and increased risk for lymph- Further, ectopic expression of LMTK3 resulted in stabilization and node metastasis after loss of its expression in primary breast further increase of HSPB8 protein levels, independently of cancer;32 (vi) INPP4B tumor suppressor protein that is frequently treatments (Figure 4b), demonstrating the effects of LMTK3 on lost in primary human breast carcinomas and is associated with HSPB8 translation. high clinical grade and tumor size;33,34 (vii) SGK3, which is linked to As HSPB8 has been shown to support proliferation of Tam- estrogen receptor both in breast cancer cell lines and in primary sensitive cell lines by inhibiting autophagy, we investigated the tumor samples, and it has been shown that its overexpression effects of LMTK3 overexpression on the survival of MCF7 cells in partially protects MCF-7 cells against apoptosis caused by the presence and absence of Tam. Tam alone resulted in B70% anti-estrogens.35,36 cell death after 6 days of treatment, whereas overexpression of LMTK3 partly rescued cells from Tam-induced cell death Validation of microarray data (Figure 4c), implying that LMTK3 is relevant for cellular survival. To clarify whether LMTK3 is able to protect from cell death by To validate our microarray results, we performed RT–qPCR analysis blocking autophagy through increased HSPB8 protein levels, we on a large subset of differentially expressed genes (Figure 3a), and examined the expression of MAPLC3, a well-known autophago- were able to reproduce the inhibitory effects of LMTK3 silencing somal marker. Immunofluorescence of MCF7 cells revealed the on their expression after E2 treatment. In addition, to further characteristic punctuate pattern of MAPLC3 after Tam treatment, validate that LMTK3 is responsible for the observed results, we whereas ectopic expression of LMTK3 seems to decrease the examined the relative mRNA levels of those genes after over- number of autophagic cells (Figure 4d). Taken together, these expressing a plasmid encoding for full-length LMTK3. As expected, results suggest that LMTK3 can contribute to the development of overexpression of LMTK3 (Figure 3b) had the opposite effects on Tam resistance via different signaling pathways including those the transcriptional levels of these genes, compared with siLMTK3, implicated in autophagic processes. confirming the specificity of LMTK3.

LMTK3 increases HSPB8 protein levels and protects MCF7 cells LMTK3 cfDNA levels in plasma and breast tumors predict from Tam-induced cell death by reducing autophagy endocrine resistance One gene of note among those signiﬁcantly modulated by LMTK3 In patients with ERa-positive breast tumors treated with aroma- was HSPB8, which has been recently described to have an tase inhibitors, LMTK3 levels determined by immunohistochem- important role in Tam resistance by inhibiting autophagy and cell istry were higher in non-responders than responders determined death.11 To determine whether LMTK3 is also able to affect HSPB8 by the changes of Ki67 labeling index changes12,13 at baseline at translational level, we silenced and overexpressed LMTK3 in the (Po0.001) (Supplementary Figure 3 and Supplementary Table 1), presence of various treatments and examined the protein but no signiﬁcant changes were detected in LMTK3 levels before

& 2013 Macmillan Publishers Limited Oncogene (2013) 3371 – 3380 LMTK3 is implicated in endocrine resistance J Stebbing et al 3376

Figure 3. Effects of LMTK3 overexpression on the transcriptional levels of selected genes. BT474 cells were transfected with (a) siControl or siLMTK3 for 48 h, or (b) empty vector (pCMV6) or full-length LMTK3 (pCMV6-LMTK3) for 24 h, followed by treatment with ETOH (vehicle) or E2 (10 nM) for 24 h. The transcriptional levels of various genes (HSPB8, RARA, MYBL1, CXL12, IGFBP4, SIAH2, MYC and LMTK3) were analyzed by RT–qPCR (*Po0.05, **Po0.01, ***Po0.0001).

and after the therapy between responders and non-responders (Po0.0001) in both LMTK3 and ESR1 were detected specifically (Table 3). There was also no correlation between baseline LMTK3 in the second cfDNA samples of the patients who relapsed again and Ki67 levels (P ¼ 0.057). Furthermore, LMTK3 levels did not suggesting acquired resistance. These samples were taken just change significantly during treatment, supporting a role in innate before relapse. resistance. Using copy number (CN) values of 42.5 and o1.5 as thresholds for amplification and deletion, respectively, there were no CN DISCUSSION variations in the LMTK3 gene identified in any normal leukocyte Endocrine resistance in breast cancer is frequent, resulting in DNA samples of the 50 patients on follow-up or in paired significant mortality. Several studies thus far have revealed various leukocytes and cfDNA of 8 healthy female controls. However, the signaling pathways implicated in this process but exact mechan- 3’- (distal) end of the LMTK3 gene was amplified in 26% of primary isms require further elucidation, especially if we are to reverse this tumor DNA samples and the entire gene was amplified in 10%. in the clinic.38–42 Having recently identified LMTK3 as a novel This tumor-specific amplification was detected in 64% of the first regulator of ERa, we wished to examine its contribution in Tam cfDNA samples and 68% of second cfDNA samples taken an re-sensitization. For this reason, we generated a Tam-resistant average of 6 and 9 years after surgery and treatment xenograft breast cancer mouse model, one we think that has (Supplementary Figure 4), suggesting a role in acquired resistance. broader utility as an endocrine-resistant model, and demonstrated A small percentage of cfDNA samples (4% of first and 2% of that inhibition of LMTK3 expression is able to restore the inhibitory second samples) also showed deletion at the 50-end of the LMTK3 tumor growth effects of Tam. As this is the first time that LMTK3 gene. Twenty five of the patients on follow-up received Tam and has been linked to the development of resistance, we performed a six of these patients went on to relapse. When results were human genome microarray analysis to identify target genes and compared, based on patients who relapsed compared with the signaling pathways affected by LMTK3 under different treatments patients who did not, significant CN gain (amplification) (E2 and Tam). As expected, several of the newly identified LMTK3-

Oncogene (2013) 3371 – 3380 & 2013 Macmillan Publishers Limited LMTK3 is implicated in endocrine resistance J Stebbing et al 3377

Figure 4. Effects of LMTK3 on MCF7 and BT474 cells. (a) Western blot of BT474 cells transfected with siControl or siLMTK3 for 72 h, or (b) with empty vector (pCMV6) or full-length LMTK3 (pCMV6-LMTK3), followed by treatment with ETOH (vehicle), E2 (10 nM) or Tam (100 nM) for 24 h. (c) Cell proliferation of MCF7 cells transfected with empty vector (pCMV6) or full-length LMTK3 (pCMV6-LMTK3) and treated with Tam. *Po0.05 (Student0s t-test). (d) Immunoﬂuorescence images of MAPLC3 autophagy marker staining in MCF7 cells transfected with empty vector (pCMV6) or full-length LMTK3 (pCMV6-LMTK3) and treated with Tam (1 mM) for 72 h.

Table 3. (a) Top. Independent-samples t-test was used for comparing baseline LMTK3 between pathological responder group and non-responder group, and for comparing baseline LMTK3 between Ki-67 responder group and non-responder group. (b) Lower. Paired-samples t-test were used for testing the change of LMTK3 after AI treatment in pathological responder or non-responder groups, and after aromatase inhibition in Ki67 responder and non-responder groups

Group N Baseline LMTK3 value P

Pathological responder 5 85.6±25.3 o0.001> Non-responder 15 200.3±12.1

Ki-67 responder 9 165.3±28.8 0.725 Ki-67 non-responder 11 176.8±17.1

Pathological Response N state LMTK3 value P

Pre-treatment 200.3±12.1 Non-responder 15 Post-treatment 161.0±15.2 0.087

Pre-treatment 85.6±24.8 Responder 5 Post-treatment 89.4±25.3 0.878

Pre-treatment 176.8±17.1 Non-responder 11 Post-treatment 162.7±17.1 0.487

Pre-treatment 165.3±28.8 Responder 9 Post-treatment 119.1±23.2 0.167 regulated genes have been previously linked to breast cancer E2-regulated gene and its expression to be upregulated in development and also to Tam resistance, while others are new. Of breast cancer,37,45,46 its potential involvement in Tam resistance note, the UGT2B15 and related UGT2B17 genes at 4q13.2 have was only recently described.11 Overexpression of LMTK3 resulted been shown to be upregulated by E2 in MCF-7 cells43 and may in an increase of HSBP8 at both the mRNA and protein levels, have a role in maintaining normal steroid hormone homeostasis suggesting a dual mechanism of action of LMTK3 on HSPB8 and preventing excessive estrogen signaling. These genes also stability. Interestingly, ectopic expression of LMTK3 partly rescued showed CNV in our SNP 6.0 study.14 proliferation of MCF7 cells (Tam-sensitive) by reducing autophagy, Among the genes whose expression was signiﬁcantly reduced via increased HSPB8 levels. These data reveal LMTK3 as a new after inhibition of LMTK3 was HSPB8 (heat shock 22kDa protein 8), regulator of HSPB8 and propose a potential involvement of LMTK3 which is a member of the small heat-shock protein superfamily.44 in the autophagic cell degradation process. However, taking into Although HSPB8 has been previously described to be an consideration the LMTK3-regulated gene-expression signature

& 2013 Macmillan Publishers Limited Oncogene (2013) 3371 – 3380 LMTK3 is implicated in endocrine resistance J Stebbing et al 3378 from our validated microarray, it is clear that further work will be Laboratories, Indianapolis, IN, USA) between 7–8 weeks of age. BT474 cells required to establish LMTK3 interaction partners. were cultured in media containing FBS and injected into the mammary fat There is very little questioning of the success of Tam and, pad of mice at a concentration of 1 Â 107 cells/injection (Champions despite meteoric rises in health-care costs for targeted therapies, Biotechnology, Baltimore, MD, USA). Pre-study tumor volumes were recorded for the experiment. When tumors reached an approximate area Tam is a readily affordable drug that has saved millions of 3 0 of 100–200 mm , animals (n ¼ 10) were randomly assigned to different women s lives. However, because not all ERa-positive breast groups and treated with i) 10 mg of control siRNA (in PBS), ii) 10 mgof cancers respond to Tam treatment, the development of methods LMTK3 siRNA, iii) 0.5 mg/kg of Tam þ 10 mg of control siRNA and, iv) to improve targeting Tam to treat the ‘right’ tumors would be of 0.5 mg/kg of Tam þ 10 mg of LMTK3 siRNA); animals were tagged and 47 enormous value and LMTK3 as a biomarker has potential here. followed individually throughout the experiment. Four intra-tumoral Research has identified two forms of resistance to Tam therapy: injections into mammary fat pad were repeated every 3 days and mice intrinsic (de novo) resistance, when ERa-positive tumors do not were terminated 3 days after the last injection. Tumor growth was respond to Tam at the outset of treatment which is uncommon, monitored using calliper measurements. At termination, primary tumors and acquired resistance, when ERa-positive tumors that initially were excised, weighed and formalin fixed. Statistical differences in tumor respond to Tam subsequently exploit the Tam-ERa complexes as a volume were determined using a two-tailed one-way (ANOVA) followed by the Dunnett’s multiple comparisons test comparing treated single-agent stimulatory growth signal or, as we and others have shown, groups (LMTK3 siRNA, or Tam) with control and combination (LMTK3 phosphorylation leads to ligand-independent activity. We have siRNA þ Tam) with standard agent (LMTK3 siRNA or Tam). previously demonstrated in a very large European and a smaller Asian cohort that high baseline levels of LMTK3 are associated with more aggressive breast tumors, and that these are those Illumina microarray hybridization 10,47 siRNA: two different siRNAs (20 nM) for LMTK3 (LMTK3 siRNA 1 (targeting patients more likely to relapse. As well as being a poor 0 0 prognostic feature, LMTK3 levels were predictive, indicating a sequence: 5 -TGTCTGCGTAACCGCACGGG-3 ) and LMTK3 siRNA 2 (targeting sequence: 5’-GTTCATCTCGGAAGCACA-3’ (Qiagen, Hilden, Germany). The poorer response to Tam. We now show that these levels are All Stars Negative Control siRNA (Qiagen) (#1027280) was used as control. generally unaltered throughout therapy, implicating LMTK3 in the BT474 cells were transfected for 48 h with the LMTK3 siRNAs or control less common form of ‘unresponsiveness’ to therapy, intrinsic siRNA using Hiperfect followed by media change and treatment with E2 resistance. (10 nM) and/or Tam (100 nM) for 24 h. All siRNA transfections as well as all Plasma cfDNA analysis of breast cancer patients showed that compound treatments were performed as three independent replicates. many of them have specific CN changes in the LMTK3 gene RNA was extracted with the RNeasy Mini Kit (Qiagen), followed by detectable in cfDNA, which mirror those in their primary tumor, digestion of genomic DNA using the RNase-free DNase-Set (Qiagen). RNA and that these persist over a number of years, despite the patient concentration was measured on Nanodrop (Peqlab, Southampton, UK). having received adjuvant therapy, and support a role for LMTK3 in RNA quality was determined on an Agilent Bioanalyzer. RNA Integrity Number for all total RNA samples was between 9.4–10 indicating excellent intrinsic endocrine resistance. Furthermore, these data for LMTK3 RNA quality. RNA labeling was performed on a Hamilton Star Roboter support the genome-wide CN analysis, which revealed the (Hamilton, Bonaduz, Switzerland) using the Illumina Total Prep-96 RNA persistence of tumor-associated CNVs in paired cfDNA samples Amplification kit from Ambion (Applied Biosystems, Carlsbad, CA, USA, years after surgery and treatment despite the fact that patients #4393543). Briefly, from 500 ng total RNA first and second strand cDNA was had no clinically evident recurrent disease.14 However, of note, synthesized, and cDNA was cleaned up using Agencourt magnetic beads significant amplification (Po0.0001) of both ESR1 and LMTK3 CleanKit (#000494, Beckman Coulter, London, UK). After in vitro transcrip- genes detected in the cfDNA of six patients who relapsed while tion, biotin-labeled cRNA was purified using Agencourt magnetic beads receiving Tam and may suggest one potential mechanism for CleanKit. cRNA concentration was determined with Quant-iT RiboGreen development of acquired resistance to Tam (and endocrine (R-11490, Invitrogen). 1,5 mg CRNA was mixed with the hybridization reagents and hybridized to Illumina Human HT-12 v4 BeadChips. All washing and therapy in general), although numbers here are low and should staining steps were performed with a Little Dipper Processor for Illumina be prospectively validated. In the adjuvant setting, it could be that BeadChips from Scigene (Sunnyvale, CA, USA). Biotin was detected with micrometastatic tumor cells are under selective pressure to harbor streptavidin-Cy3 (#PA43001, Amersham Biosciences, Piscataway, NJ, USA). mutations that drive resistance to the therapy being applied and The BeadChips were scanned on a Illumina Bead Array Reader (San Diego, to later relapse. Amplifications in ESR1 and LMTK3 are two possible CA, USA). milestones on this path to relapse. In aggregate, these data support the conclusion that LMTK3 is Bioinformatic analysis relevant in the development of endocrine resistance in breast BeadChip raw data output contains the average signal intensity and the cancer. detection P-value for each probe. Analyses were performed using GeneData’s Expression Analyst (version 2.2.9). For quality control box plots of signal intensity, unsupervised hierarchical clustering and Principal MATERIALS AND METHODS Components Analysis for both un-normalized and median-normalized data Cell lines and arrays were carried out without identifying any outliers. The analyses revealed MCF7 and BT474 were maintained in DMEM supplemented with 10% FCS even signal distribution within the set of experiments. A three-way ANOVA (or 10% charcoal-dextran stripped serum) and 1% penicillin/streptomycin/ was used to investigate the influence of the factors ‘cell lines’, ‘siRNA-’, and ‘compound-’treatment on differential gene expression (siRNA: control, glutamine. All cells were incubated at 37 1C in humidified 5% CO2. Estradiol (E2) and Tam were obtained from Sigma and dissolved in 100% mock and siLMTK3; compound: ETOH, E2, E2 þ Tam, E2 þ ICI). Most ethanol; charcoal-dextran stripped serum was obtained from Gemini variability in gene expression could be explained by the factor cell line (Northern California, CA, USA). The following antibodies were used: LMTK3 (data not shown) whereas both kinds of treatment had comparingly much mouse monoclonal (Santa Cruz, Santa Cruz, CA, USA), MAPLC3 rabbit smaller effects. Therefore, we investigated the effects of siRNA- and polyclonal (Cell Signaling, Danvers, MA, USA), HSPB8 rabbit polyclonal (Cell compound-treatment in each cell line separately using two-way ANOVA. Signaling), FLAG mouse monoclonal (Cell Signaling), b-actin mouse Significantly differential gene expression between any two groups (as monoclonal (Abcam, Cambridge, UK) The full-length human LMTK3 defined by the combination of siRNA and compound treatment) due to complementary DNA (cDNA) sequence (NM_001080434) was cloned in compound treatment was identified in a two-way ANOVA using BT474 cell the eukaryotic expression vector pCMV6 (Myc/Flag tagged), using the SgfI line (factors: siRNA- and compound-treatment; effect: compound treat- and MluI restriction enzymes. ment, or siRNA treatment, or the interaction thereof). The attached Excel table lists the 4718 genes showing significant changes in gene expression between any two groups (BH-Qo0.05) To look Xenograft model for synergistic effects, we calculated mean fold changes in expression of We compared vehicle, LMTK3 siRNA, Tam, and a combination of LMTK3 these genes due to Tam inhibition (siCTRL/E2 versus siCTRL/E2 þ Tam) or siRNA and Tam in BT474 cells in immuno-compromised SCID mice (Harlan LMTK3 depletion (siCTRL/E2 versus siLMTK3/E2) or the combined effect

Oncogene (2013) 3371 – 3380 & 2013 Macmillan Publishers Limited LMTK3 is implicated in endocrine resistance J Stebbing et al 3379 (siCTRL/E2 versus siLMTK3/E2 þ Tam). Synergistic effects were deﬁned UICC Yamagiwa-Yoshida Memorial International Cancer Study Grant to GG. We thank according to the following formula: fc(siCTRL/E2 versus siLMTK3/E2 þ R & E Girling and friends, Cancer Research UK and the Pink Ribbon Foundation for Tam)4fc(siCTRL/E2 versus siCTRL/E2 þ Tam) *fc(siCTRL/E2 versus siLMTK3/E2). their support. This work was also supported by the Imperial BRC and ECMC.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene (2013) 3371 – 3380 & 2013 Macmillan Publishers Limited