Association of tamoxifen resistance and lipid reprogramming in breast cancer

Background Tamoxifen treatment of estrogen receptor (ER)-positive breast cancer reduces mortality by 31%. However, over half of advanced ER-positive breast cancers are intrinsically resistant to tamoxifen and about 40% will acquire the resistance during the treatment. Methods In order to explore mechanisms underlying endocrine therapy resistance in breast cancer and to identify new therapeutic opportunities, we created tamoxifen-resistant breast cancer cell lines that represent the luminal A or the luminal B. Gene expression patterns revealed by RNA-sequencing in seven tamoxifen-resistant variants were compared with their isogenic parental cells. We further examined those transcriptomic alterations in a publicly available patient cohort. Results We show that tamoxifen resistance cannot simply be explained by altered expression of individual genes, common mechanism across all resistant variants, or the appearance of new fusion genes. Instead, the resistant cell lines shared altered gene expression patterns associated with cell cycle, protein modification and metabolism, especially with the cholesterol pathway. In the tamoxifen-resistant T-47D cell variants we observed a striking increase of neutral lipids in lipid droplets as well as an accumulation of free cholesterol in the lysosomes. Tamoxifen-resistant cells were also less prone to lysosomal membrane permeabilization (LMP) and not vulnerable to compounds targeting the lipid metabolism. However, the cells were sensitive to disulfiram, LCS-1, and dasatinib. Conclusion Altogether, our findings highlight a major role of LMP prevention in tamoxifen resistance, and suggest novel drug vulnerabilities associated with this phenotype. Electronic supplementary material The online version of this article (10.1186/s12885-018-4757-z) contains supplementary material, which is available to authorized users.


Background 13
Tamoxifen treatment of estrogen receptor (ER)-positive breast cancer reduces mortality by 31%. However, 14 over half of advanced ER-positive breast cancers are intrinsically resistant to tamoxifen and about 40% 15 will acquire the resistance during the treatment. 16

Methods 17
In order to explore mechanisms underlying endocrine therapy resistance in breast cancer and to identify 18 new therapeutic opportunities, we created tamoxifen-resistant breast cancer cell lines that represent the 19 luminal A or the luminal B. Gene expression patterns revealed by RNA-sequencing in seven tamoxifen-20 resistant variants were compared with their isogenic parental cells. We further examined those 21 transcriptomic alterations in a publicly available patient cohort. 22

Results 23
We show that tamoxifen resistance cannot simply be explained by altered expression of individual genes, 24 common mechanism across all resistant variants, or the appearance of new fusion genes. Instead, the 25 resistant cell lines shared altered gene expression patterns associated with cell cycle, protein modification 26 and metabolism, especially with the cholesterol pathway. In the tamoxifen-resistant T-47D cell variants 27 we observed a striking increase of neutral lipids in lipid droplets as well as an accumulation of free 28 cholesterol in the lysosomes. Tamoxifen-resistant cells were also less prone to lysosomal membrane 29 permeabilization (LMP) and not vulnerable to compounds targeting the lipid metabolism. However, the 30 cells were sensitive to disulfiram, LCS-1, and dasatinib. 31

Conclusion 32
Altogether, our findings highlight a major role of LMP prevention in tamoxifen resistance, and suggest 33 novel drug vulnerabilities associated with this phenotype. 34

Keywords 35
Tamoxifen resistance; breast cancer; lysosomal membrane permeabilization; RNA-sequencing; drug 36 sensitivity and resistance testing 37 Background 38 Approximately two thirds of breast cancers are estrogen receptor (ER) positive. As the receptor stimulates 39 proliferation of mammary epithelial cells, it is also an important target in anti-hormonal cancer therapy. 40 One of the most prescribed ER antagonists for first line therapy is tamoxifen that has helped millions of 41 women since its discovery 50 years ago [1]. However, de novo or acquired drug resistance towards 42 tamoxifen is a notable problem and the later affects approximately 40% of patients receiving tamoxifen 43 [2]. 44 In addition to its intended anti-cancer effects, tamoxifen is known to have both direct and indirect effects 45 on the cellular lipid metabolism. It has been shown to reduce blood cholesterol levels [3] and to be 46 protective against cardiovascular diseases [4]. However, approximately 43% of the patients treated with 47 tamoxifen develop hepatic steatosis, including the accumulation of neutral lipids to lipid droplets in 48 hepatic cells [5]. Tamoxifen can regulate the lipid balance e.g by binding to the microsomal antiestrogen 49 binding sites (AEBS), which are associated with cholesterol metabolism [6]. This mechanism has been 50 linked to control cell growth, differentiation and apoptosis in the presence of reactive oxygen species (ROS) 51 and has been established as another mode by which tamoxifen induces cytotoxicity [7,8]. 52 On the other hand, reprogrammed metabolism is one hallmark of cancer cells [9] and has recently been 53 suggested as a new mode of drug resistance in cancer therapy [10,11]. The metabolic intermediates can 54 supply cancer cells with membrane phospholipids, with energy through the β-oxidation pathway or with 55 pro-tumorigenic lipid-signaling molecules such as lysophosphatidic acid [12]. Some studies even 56 suggested a role of cholesterol metabolism in tamoxifen resistance [ Lysosomal membrane permeabilization (LMP) assay 146 In order to measure the integrity of lysosomal membranes, we performed the detection of damaged 147 lysosomes by galectin-1 and -3 translocation according to the previously published protocol [31]. We first 148 established that galectin-3 was in our cell lines a more reliable marker to detect its translocalization to the 149 lysosomes compared to galectin-1. We then seeded 2000 cells/well of T-47D, T-47D Tam1 and Tam2 on  150 PE Cell-Carrier 384-well plate +/-1 µM 4-OH-tamoxifen. After 72 h incubation, 1 mM LLMOe was added 151 as LMP induction control and incubated for 1 h. Cells were then fixed with 4 % PFA and stained with 152 galectin-3 detected with Alexa 568 (461nm), ceruloplasmin as cell segmentation marker detected with 153 Alexa 488 (488 nm, Additional file1) and Hoechst (405nm) for detection of the nuclei. 16 fields-of-view 154 were acquired with the two sCMOS cameras (2160x2160 pixels) containing Opera Phenix HCS system 155 (PerkinElmer) in a widefield mode with the 40x water immersion objective (NA 1.1). Exposure time and 156 laser power were kept constant for each individual staining across different cell lines and conditions. We 157 utilized the Columbus Image Data Storage and Analysis System (PerkinElmer) to analyze the multi-channel 158 images. First, the images were preprocessed to correct non-uniform illumination. Next, individual nuclei 159 were segmented from the Hoechst channel. The minimum area of a nucleus was set to 30 µm² to remove 160 the detection of debris in the image background. Starting from the detected nuclei, the segmented regions 161 were propagated to cover cell cytoplasm stained with ceruloplasmin. The cells that touched the image 162 border were discarded. Finally, spot detection was used to segment galectin-3 stained spots. The 163 maximum radius of the spots was set to 1 µm. We defined cells with more than 1 spot as galectin-3 positive 164 to exclude false positive detection. Further, we calculated the percentage of galectin-3 positive cells and 165 the average spots per cell. 166 Drug sensitivity and resistance testing (DSRT) and high-content phenotypic drug profiling 167 33 compounds that target lipid and cholesterol metabolism, or induce LMP, were selected for the DSRT 168 by literature and vendor research (Additional file 2). As in the previous DSRT screens [15,32], the dissolved 169 drugs were transferred in five different concentrations covering a 10 000-fold concentration range into 170 384-well plates in duplicates mirrored after column 12. 1500 cells of T-47D parental, Tam1, and Tam2 cells  171 were then seeded into the wells in normal growth media on columns 1-12 of each plate and in media 172 supplemented with 1 μM 4-OH-tamoxifen on columns 13-24.  file 4), providing means to obtain expression estimates between 33,600 and 37,000 genes per sample. We 205 determined differential gene expression as log2 change of >|1|, and the difference of gene expression > 206 |10| CPM between the resistant clone and its isogenic parental cell line (see Methods). Using this filtering, 207 we identified >1200 differentially expressed genes in MCF-7-and T-47D-derived cells, and <400 genes in 208 BT-474-and ZR-75-1-derived cells. On average 59 % of differentially expressed genes were upregulated in 209 the majority of resistant cell lines (Table 1). Interestingly, only about 35 % of altered genes in T-47D as 210 well as BT-474, and only 24 % in ZR-75-1 were shared between the resistant cells derived from the same 211 parental cell line (Additional figure 1A). Additionally, no common differentially expressed genes were 212 identified, highlighting that each of the cell lines had developed resistance through a distinct molecular 213 pattern ( Figure 1A  fold increase (patient 1 and 3, respectively, Additional file 6). 232

Triglycerides and cholesterol esters are increased in the resistant T-47D cell lines 233
To reveal pathways associated with tamoxifen resistance, we analyzed the differentially expressed genes 234 with Enrichr [26,27]. Based on Enrichr's Reactome 2016 analysis with an adjusted p-value below 0.001, 235 we observed multiple enriched pathways in different resistant cell lines (Table 2 and Additional File 7). 236 The most striking differences were found in the T-47D Tam1 and Tam2 cells, which displayed changes in 237 metabolism associated genes, especially those involved in cholesterol and related lipid metabolism (Table  238 2, Figure 2A). 239 In addition, we observed an upregulation of genes involved in cholesterol biosynthesis in all three 240 metastatic patient samples from McBryan et al. study (Figure 2A). Therefore, we focused our studies on 241 these pathways within the T-47D cell lines (Table 2 and Additional File 7). To investigate whether 242 deregulation of genes involved in cholesterol biosynthesis could affect the cellular cholesterol balance, 243 we stained cellular free cholesterol with filipin, a fluorescent cholesterol-binding compound. Notably, we 244 observed increased intracellular amounts of free cholesterol in the resistant cells, displaying a cumulus 245 cloud-like staining pattern ( Figure 2B). To quantify the presence of major cellular lipid species e.g. 246 cholesterol, cholesterol esters, and triglycerides, their amounts were further determined with thin layer 247 chromatography. The total cellular free cholesterol remained unchanged, suggesting that only the 248 distribution of free cholesterol was altered in the resistant cells. However, we observed an increase in 249 neutral lipids (cholesterol esters and triglycerides) upon tamoxifen treatment. The increase in triglycerides 250 was significantly high (4 to 7 fold-increase) in resistant cells compared to parental cells ( Figure 2C). To 251 visualize the changes in neutral lipid amounts as well as their intracellular distribution, we stained the 252 cells with LipidToxGreen, a fluorescent dye binding specifically to neutral lipids. The analysis indicated that 253 most of the neutral lipids accumulated in enlarged lipid droplets, which fill the cytoplasm ( Figure 2D). 254 Moreover, RNA sequencing results implicated that the expression of Peroxisome Proliferator-Activated 255 Receptor gamma (PPARG), which is known to regulate several lipid droplet proteins, was upregulated in 256 resistant cells. In addition, the ATP Binding Cassette Subfamily A Member 1 (ABCA1), which functions as a 257 cholesterol efflux pump was downregulated (Additional file 6). suggests that circumvention of LMP in the resistant cells leads not only to tamoxifen resistance but may 283 also decrease their sensitivity to other drugs. 284

Drug testing of tamoxifen resistant cells reveals sensitivity to dasatinib, disulfiram and LCS-1 285
Guided by our RNA-sequencing results we selected 33 drugs, known to affect the genes or pathways 286 involved in lysosomal alterations and lipid metabolism as well as some drugs identified in our previous 287 screen (Additional file 2, [15]). As readouts for the DSRT, we applied both enzymatic cell viability 288 measurement (CTG) as well as a phenotypic image-based analysis using LipidToxGreen to observe neutral 289 lipids in lipid droplets together with Hoechst to detect nuclei. 290 The cell viability measurement revealed drugs that reduced ATP levels in tamoxifen-resistant cells similarly  291 to the control cells, independently of their lipid accumulation phenotype ( Figure 4A, Additional file 8). 292 Dasatinib, a dual Abl/Src inhibitor was more effective in killing the tamoxifen resistant cell lines compared 293 with the parental cells in agreement with our previous results [15]. Tamoxifen-resistant cells were more  294 sensitive to microtubule depolymerizing drugs, such as vincristine and vinorelbine, when measured by 295 ATP amounts. Interestingly, the T-47D Tam2 cells were especially sensitive to vinorelbine induced 296 cytotoxicity ( Figure 4A, Additional file 8). The mitotic inhibitors paclitaxel and docetaxel (microtubule 297 stabilizers) were less effective in the T-47D Tam1 cells. (Figure 4A, Additional file 8). 298 The most effective drug against all the T-47D clones was disulfiram, a specific inhibitor of aldehyde-299 dehydrogenase (ALDH1). All the clones responded to AZD8055, a dual mTOR inhibitor, although T-47D 300 Tam1 and Tam2 showed reduced sensitivity. Atorvastatin, which inhibits HMGCoA reductase, did not 301 affect the CTG DSS levels ( Figure 4A) but was able to reduce the cell count in the parental and even more 302 in the T-47D Tam2 cells ( Figure 4B). The SOD-1 inhibitor LCS-1 was effectively killing both parental and 303 resistant T-47D clones, and RSL-3, a ferroptosis activator due to inhibition of glutathione peroxidase 4, 304 induced cell death in all the cell lines, with somewhat reduced response in T-47D Tam1 ( Figure 4B, 305 Additional file 8).

306
To see whether any of the compounds are able to revert the lipid phenotype prior to reducing the cell 307 viability (ATP-measurement) or induction of cell death (cell count), we specifically monitored the changes 308 in neutral lipids by quantifying the average LipidToxGreen intensity per well (Additional file7). The 309 measured significant increase in the intensity was within the 2-fold range, and we were able to confirm 310 the trends from the biochemical screen where we observed a neutral lipid accumulation in the resistant 311 cell lines ( Figure 2C, Figure 4C). Whereas most of the drugs had minor effects on the LipidToxGreen 312 intensity (Additional file 9), the LXR-agonist TO901317 increased the lipid phenotype most strikingly in the 313 parental cells, with less increase particularly in T-47D Tam1 cells ( Figure 4D). In addition, methyl--314 cyclodextrin, a membrane cholesterol-depleting agent, caused a lipid droplet accumulation phenotype, 315 mostly in the parental cell line prior to cell killing in the highest concentration (Additional file 9). 316 317 Discussion 318 In this study, we utilized RNA-sequencing and pathway analysis to understand the underlying tamoxifen 319 resistance and identify resistance-specific drug vulnerabilities. We revealed the involvement of lipid 320 metabolism in tamoxifen resistance as well as pointed out potential therapeutic ways to target these 321 pathways. 322 Gene expression analysis on tamoxifen-resistant cells reinforced our previous finding on breast cancer 323 cells using a variety of molecular pathways as they acquire tamoxifen resistance [15]. The difference in 324 gene expression was reflected in the scale and scope of differentially expressed genes, and in the lack of 325 shared genes across all the cell lines ( Figure 1). In agreement with this finding, the only study that has 326 performed sequential tumor transcriptome analysis on patients developing endocrine resistance, also 327 identified less than 3% of differentially expressed genes across patients ( Figure 1C [16]). Despite the 328 overall transcriptome profiles being distinct across the resistant cell lines, we were able to identify five 329 genes that were concordantly differentially expressed in the luminal A subtype resistant cells (Additional 330 figure 1B). Of these, SERPINA1, encoding for a serine protease inhibitor primarily targeting elastase, is 331 known to bind ER in a 17β-estradiol (E2) -independent manner, which leads to an increase in its 332 expression [39]. Therefore the observed expression changes could be due to the down-and upregulation 333 of ER in these cell lines [15]. Interestingly, in all three metastatic samples from the McBryan et al. study, 334 we observed an increase in SERPINA1, which is accompanied by a slight increase of ESR1 transcription 335 (Additional file 6). Pathway analysis of the differentially expressed genes identified several paths involved 336 in acquired tamoxifen resistance (Table 2, Figure 2A). 337 In this study, we investigated the tamoxifen-induced changes observed in lipid metabolism, which 338 occurred in the T-47D tamoxifen-resistant cell lines (Table 2, Figure 2). We also made the equivalent 339 finding in a patient's metastatic tissue (Figure 2A) Tam1 and Tam2 were even more resistant towards LMP ( Figure 3C and D), showing that tamoxifen can 351 hinder it, and in acquired resistance, this phenomenon is even more prominent. Thus, impeded lysosomal 352 membrane permeabilization may additionally enhance the co-resistance to other cancer drugs during 353 acquired tamoxifen resistance. 354 Reducing the reactive oxygen species (ROS) is another mechanism by which cells avoid lysosomal induced 355 cell death [44]. We speculate that resistant T-47D cells are able to reduce oxidative stress by upregulation 356 of SOD1 (Additional file 6) and may therefore be less sensitive to lysosomal cell death. This hypothesis is 357 further supported by the fact that the resistant cells were highly sensitive to the SOD1 inhibitor LCS-1. The 358 capability of erastin to activate ferroptosis is instead inhibited by antioxidants, and it was more effective 359 in parental than in resistant cells. The ferroptosis activator RSL-3, which inhibits the glutathione 360 peroxidase 4, an enzyme that protects from oxygen damage, induced cell death in all the cell lines ( Figure  361 4 and Additional file 8). This further supports the assumption that the T-47D cells are able to reduce 362 oxidative stress and are therefore less sensitive to lysosomal cell death. 363 Disulfiram, which targets ALDH1 to increase oxidative stress, was highly effective in both parental and 364 tamoxifen-resistant T-47D cell clones (Figure 4 and Additional file 8). The effectiveness of disulfiram is 365 currently investigated in metastatic breast cancer in a phase II clinical trial [46]. ALDH2, another target of 366 disulfiram, is upregulated in T-47D Tam1 but not in Tam2 (Additional file 6). High levels of ALDH1 have 367 been shown to predict resistance in women treated with tamoxifen [47], but as ALDH1A1 is expressed at 368 very low levels in the T-47D cell lines (Additional file 6), we assume that the sensitivity to disulfiram could 369 be due to its capability to disable antioxidation mechanisms of the cells [48]. to be more resistant to chemotherapy [51]. We found over 3-fold upregulation of stearoyl-CoA desaturase 377 (SCD), encoding for a rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids, in the 378 tamoxifen-resistant T-47Ds (Additional file 6). Whether it alone is able to induce the increase in TGs, 379 remains to be investigated. In line with this speculation, SCD overexpression has been observed, in 380 different cell types as well as in tamoxifen-induced hepatocyte steatosis, to significantly increase the rate 381 of triglyceride synthesis [52]. The compounds directly affecting lipid metabolism, such as C75, Bezafibrate, 382 T 0070907, TO901317, and Orlistat, had no or only little effect on cell viability or the lipid phenotype 383 (Figure 4 and Additional files 8, 9). This suggests that the T-47D cells are able to compensate the drug-384 induced lipid imbalance with several mechanisms, which would be compelling to study in depth. 385

Conclusion 386
Taken together, our results highlight that tamoxifen resistant cell lines can potentially be used as a 387 representative model for studies of tamoxifen-resistant patients. We propose that the breast cancer cells 388 can acquire tamoxifen resistance by dysregulation of different cellular pathways, dependent on their 389 individual molecular phenotypes. Here, we highlight the inhibition of lysosomal membrane 390 permeabilization as one of the mechanisms to avoid cell death, whereas an increase in neutral lipids may 391 enable the further survival of these cells. We further propose that drugs targeting cellular antioxidation 392 machinery may be able to overcome tamoxifen resistance. However, investigating the relevance of the 393 proposed mechanism of acquired resistance in patients remains a challenge.  and Lamp2 (B), detecting the lysosomal-associated-membrane-proteins 1 and 2 +/-1µM 4-OH-tamoxifen. 586 Tamoxifen-resistant cells are less sensitive to lysosomal membrane premeabilization detected with  587 galectin-3 (orange) translocation (C, images were differently enhanced for visualization purposes) and 588 measurement of galectin-3 positive cells (D upper graph) as well as number of galectin-spots per cell (D 589 lower image). Galectin-3 measurements were done on the raw image. Only significant differences (p-value 590 <0.05) between the same clone as well as of the comparison between resistant and tamoxifen-resistant 591 cells in the same treatment conditions are indicated all other comparisons can be found in Additional File 592 3. E Mature cathepsin D is downregulated in tamoxifen resistant cells. 593   ** ** ** ** ** ** **