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. Author manuscript; available in PMC: 2021 Oct 30.
Published in final edited form as: Anticancer Res. 2021 Feb;41(2):583–599. doi: 10.21873/anticanres.14810

Acquisition of Letrozole Resistance Through Activation of the p38/MAPK Signaling Cascade

RASHIDRA R WALKER 1, KAREN M GALLEGOS 1, MELYSSA R BRATTON 2, KITANI P LEMIEUX 3, KUN ZHANG 4, GUANGDI WANG 5, A MICHAEL DAVIDSON 1, SYREETA L TILGHMAN 1
PMCID: PMC8556662  NIHMSID: NIHMS1748519  PMID: 33517263

Abstract

Background/Aim:

Previous reports identified a global proteomic signature of estrogen-independent letrozole resistant breast cancer cells, however, it remains unclear how letrozole-resistance is impacted when cells remain estrogen receptor positive (ER+).

Materials and Methods:

To capture the protein expression profile associated with ER+ Aromatase inhibitor (AI) resistance, a global proteomic analysis was conducted using the letrozole-sensitive (T47Darom cells) and letrozole-resistant cells (T47DaromLR cells). To examine the molecular features associated with this phenotype Kaplan–Meier analysis, phospho-antibody arrays, proliferation and apoptosis assays were conducted.

Results:

MAP3K6 was up-regulated in the T47DaromLR cells by 3.2-fold (p<0.01) which was associated with a decrease in relapse-free survival among breast cancer patients (p=0.0019). Members of the MAPK/p38 pathway (i.e., phospho-MKK6, phospho-p38, phospho-RSK1, phospho-RSK2, and p70S6K MAPK) were also increased in the T47DaromLR cells, while inhibiting p38 led to decreased proliferation and induction of apoptosis.

Conclusion:

Activation of the p38/MAPK pathway leads to ER+ AI-resistance.

Keywords: Breast cancer, letrozole, aromatase inhibitor, resistance, p38, MAPK

Aromatase inhibitors, like letrozole, are the first-line therapy for postmenopausal women with estrogen-dependent breast cancer. These agents block the biosynthesis of estrogen through aromatase inhibition and inhibit the proliferation of estrogen-dependent breast tumors. Unfortunately, after several years some patients develop therapeutic resistance, and the tumors continue to proliferate. To study aromatase inhibitor resistance in the laboratory setting, a letrozole resistant cell line was developed by inoculating the MCF-7Ca cells (MCF-7 cells stably transfected into the human aromatase gene) into immunocompromised mice and treating the mice with letrozole for 56 weeks (1). After long-term treatment, the tumors acquired resistance to letrozole, were cultured in vitro and termed long term letrozole treated (LTLT)-Ca cells. Previous studies in our lab have demonstrated that as the letrozole-sensitive cells (AC-1) transition to the letrozole-resistant phenotype (LTLT-Ca) they are associated with estrogen independence, up-regulation of growth factor signaling cascades, and increased motility (2). However, the changes that occur as cells acquire resistance to letrozole while maintaining estrogen dependence remains unclear.

To this end, an in vitro model of letrozole resistance was developed whereby the T47D parental cell line was stably transfected with the human aromatase gene and termed the T47Darom cells. The T47Darom cells were treated with letrozole for 75 weeks until they became resistant to letrozole and were called T47DaromLR cells. When the T47DaromLR cells were characterized, they retained both aromatase activity and ER expression (3). As such, this was an ideal model to test the hypothesis that there are distinct proteomic profiles between estrogen-dependent and estrogen-independent letrozole resistant breast cancer cells, which may be useful for therapeutic decision making. Here, we examined the proteomic signature associated with T47D cells as they acquire resistance to an aromatase inhibitor.

Materials and Methods

Cell culture.

In this study, we utilized two cell lines previously developed and derived from the T47D parental breast cancer cell line (3). The T47Darom cells (the T47D cells stably transfected with the human aromatase gene) were cultured and maintained in phenol red DMEM media (Invitrogen, Waltham, MA, USA) supplemented with 5% fetal bovine serum (FBS), penicillin-streptomycin, antimycotic-antibiotic (10,000 U/ml penicillin G sodium; 10,000 mg/ml streptomycin sulfate), and 0.75 mg/ml geneticin (Invitrogen). The T47DaromLR cells were generated by treating the T47Darom for 75 weeks in the presence of 10 μM letrozole and were maintained in phenol red-free DMEM media (Invitrogen) supplemented with 10% charcoal-stripped fetal bovine serum (FBS), penicillin-streptomycin, antimycotic-antibiotic (10,000 U/ml penicillin G sodium; 10,000 mg/ml streptomycin sulfate), 7.5 mg/ml geneticin (Invitrogen), and 1 mM letrozole (Sigma-Aldrich, St. Louis, MO, USA). The cells were maintained in a tissue culture incubator in a humidified atmosphere of 5% CO2 and 95% air at 37°C. Both cell lines were a generous gift from ITT Research Institute.

MAPK and RTK phosphorylation antibody arrays.

To compare the changes in activation/phosphorylation of MAPK and Receptor Tyrosine Kinases (RTK) between the T47Darom and T47DaromLR cells, the Human MAPK Phosphorylation Antibody Array (ab211061, Abcam, Cambridge, MA, USA) and the Human RTK Phosphorylation Antibody Array Membrane (ab193662, Abcam), were evaluated respectively. The antibody arrays consisted of nitrocellulose membranes containing anti-MAPK or anti-RTK antibodies spotted duplicate and include positive and negative controls and a blank. Cell lysates were prepared from T47Darom and T47DaromLR cells using Cell Lysis Buffer supplemented with Phosphatase Inhibitor and Protease Inhibitor Cocktail and stored at −80°C until use. For each cell lysate, 900 μg of total protein were determined by the Bradford Assay (BioRad, Hercules, CA, USA) and incubated in 2 ml of 1X Blocking Buffer at room temperature for 30 min. The antibody array membranes were washed and subsequently incubated with Detection Antibody Cocktail (for MAPK assays) or with biotinylated anti-phosphotyrosine antibody (for RTK assays) overnight at °C to detect phosphorylated cytokines or tyrosine on activated receptors. After washing and incubation with HRP-streptavidin, the membranes were subjected to visualization with chemiluminescence-based detection method.

Cell lysis.

T47Darom and T47DaromLR control cells were cultured to 80% confluence in standard growth medium as described above and washed three times with cold Hank’s Buffered Salt Solution (HBSS), then collected with a cell scraper. Nonidet P-40 cell lysis buffer (Invitrogen) containing an additional 1 mM of phenylmethyl sulfonyl fluoride (PMSF) and protease inhibitor mixture (Sigma-Aldrich) was used to extract total cellular proteins. The concentration of proteins was measured with BCA assay (Pierce Biotechnology, Rockford, IL, USA). The cell lysates were stored at –80°C before further processing.

Trypsin digestion.

Protein samples were digested with sequencing grade modified trypsin (Promega Corp, Madison, WI, USA) according to the manufacturer’s instructions. Briefly, to aliquots of 100 μg of protein sample were added 45 μl of 200 mM triethylammonium bicarbonate (TEAB) and the final volume was adjusted to 100 μl with ultrapure water. Five microliters of 200 mM Tris (2-carboxyethyl) phosphine (TCEP) were added and the resulting mixture was incubated for 1 h, then 5 μl of 375 mM iodoacetamide were added and the mixture was incubated for 30 min in the dark. After incubation, 1 ml of prechilled acetone was added and the precipitation was allowed to proceed overnight. The acetone-precipitated protein pellets were suspended with 100 μl of 200 mM TEAB and 2.5 μg of trypsin were added to digest the sample overnight at 37°C.

Tandem mass tags (TMT) labeling.

Tandem mass tags TMT6 (Thermo Scientific, Waltham, MA, USA) with different molecular weights (126~131 Da) were applied as isobaric tags for relative and absolute quantification. According to the manufacturer’s protocols, the digested samples were individually labeled with TMT6 reagents for 1 h as follows: three 100-μg aliquots of digested peptides from the T47Darom cells were each labeled with a different isobaric tag (TMT126, 127, and 128, respectively). Likewise, 100 μg aliquots of peptides from the T47DaromLR cells were labeled with TMT129, 130, and 131 mass tags, respectively. The labeling reaction was quenched with 5% hydroxylamine. Finally, the six labeled peptide aliquots were combined for subsequent fractionation.

Fractionation of labeled peptide mixture using a strong cation exchange column.

The combined TMT labeled peptide mixture was fractionated with a strong cation exchange column (SCX) (Thermo Scientific) on a Shimadzu 2010 HPLC equipped with a UV detector (Shimadzu, Columbus, MD, USA). The mobile phase consisted of buffer A (5 mM KH2PO4, 25% acetonitrile, pH 2.8) and buffer B (buffer A plus 350 mM KCl). The column was equilibrated with buffer A for 30 min before sample injection. The mobile phase gradient was set as follows at a flow rate of 1.0 ml/min: (a) 0 to 10 min: 0% buffer B; (b) 10 to 40 min: 0% to 25% buffer B, (c) 40 to 45 min: 25% to 100% buffer B; (d) 45 to 50 min: 100% buffer B; (e) 50 to 60 min: 100% to 0% buffer B; (f) 60 min to 90 min: 0% buffer B. A total of 60 fractions were initially collected, lyophilized, and combined into 15 final fractions based on SCX chromatographic peaks.

Desalination of fractionated samples.

A C18 solid-phase extraction (SPE) column (Hyper-Sep SPE Columns, Thermo-Fisher Scientific) was used to desalt all collected fractions. The combined 15 fractions were each adjusted to 1 ml final volume containing 0.25% (v/v in water) trifluoroacetic acid (TFA, Sigma-Aldrich). The C18 SPE columns were conditioned before use by filling them with 1 ml acetonitrile and allowing the solvent to pass through the column slowly (~3 min). The columns were then rinsed three times with 1 ml 0.25% (v/v in water) TFA solution. The fractions were loaded on to the top of the SPE cartridge and allowed to elute slowly. Columns were washed four times with 1-ml 0.25% TFA aliquots before the peptides were eluted with 3×400 μl of 80% acetonitrile/0.1% formic acid (aqueous).

LC-MS/MS analysis on LTQ-Orbitrap.

Peptides were analyzed on an LTQ-Orbitrap XL instrument (Thermo-Fisher Scientific) coupled to an Ultimate 3000 Dionex nanoflow LC system (Dionex, Sunnyvale, CA, USA). High mass resolution was used for peptide identification and high energy collision dissociation (HCD) was employed for reporter ion quantification. The RP-LC system consisted of a peptide Cap- rap cartridge (0.5×2 mm) (Michrom BioResources, Auburn, CA, USA) and a prepacked BioBasic C18 PicoFrit analytical column (75 μM i.d. ×15 cm length, New Objective, Woburn, MA, USA) fitted with a For-tisTip emitter tip. Samples were loaded onto the trap cartridge and washed with mobile phase A (98% H2O, 2% acetonitrile, and 0.1% formic acid) for concentration and desalting. Subsequently, peptides were eluted over 180 min from the analytical column via the trap cartridge using a linear gradient of 6–100% mobile phase B (20% H2O, 80% acetonitrile, and 0.1% formic acid) at a flow-rate of 0.3 μl per min using the following gradient: 6% B for 5 min; 6–60% B for 125 min; 60–100% B for 5 min; hold at 100% B for 5 min; 100–6% B in 2 min; hold at 6% B for 38 min.

The LTQ-Orbitrap tandem mass spectrometer was operated in a data-dependent mode. Briefly, each full MS scan (60,000 resolving power) was followed by six MS/MS scans where the three most abundant molecular ions were dynamically selected and fragmented by collision-induced dissociation (CID) using a normalized collision energy of 35%, and the same three molecular ions were also scanned three times by HCD-MS2 with collision energy of 45%. MS scans were acquired in profile mode and MS/MS scans in centroid mode. LTQ-Orbitrap settings were as follows: spray voltage 2.0 kV, 1 microscan for MS1 scans at 60,000 resolutions (fwhm at m/z 400), microscans for MS2 at 7500 resolutions (fwhm at m/z 400); full MS mass range, m/z 400–1400; MS/MS mass range, m/z 100–2000. The “FT master scan preview mode,” “Charge state screening”, “Monoisotopic precursor selection” and “Charge state rejection” were enabled so that only the 2+, 3+, and 4+ ions were selected and fragmented by CID and HCD.

Database search and TMT quantification.

The protein search algorithm used was Mascot v2.3.01 (Matrix Science, Boston, MA, USA). Mascot format files were generated by the Proteome Discoverer 1.2 software (Thermo-Fisher Scientific) using the following criteria: database, IPI_Human.fasta. v3.77 (containing 89,422 entries and concatenated with the reversed versions of all sequences.); enzyme, trypsin; maximum missed cleavages, 2; Static modifications, carbamidomethylation (+57 Da), N-terminal TMT6plex (+229 Da), lysyl TMT6plex (+229 Da). Dynamic modifications, N-terminal Clnpyro- Glu (+17 Da); methionine oxidation (+16 Da); STY phosphorylation (+80 Da); Precursor mass tolerance was set at 20 ppm; fragment match tolerance was set at 0.8 Da. Peptides reported by the search engine were accepted only if they met the false discovery rate of p<0.05 (target decoy database), a Mascot ion score ≥30 for peptide identifications was required. For TMT quantification, the ratios of TMT reporter ion abundances in MS/MS spectra generated by HCD (up to six reporter ions ranging from m/z 126.12 to m/z 131.14) from raw data sets were used to calculate fold changes in proteins between control and treatment.

Kaplan–Meier (KM) survival analysis.

The application of KM plot has been described in detail previously (4). Briefly, KM plots were obtained using the KM Plotter web-based (kmplot.com/analysis) curator, which surveys public microarray repositories for relapse-free and overall survival among patients with breast, lung, ovarian or gastric cancers. The KM Plotter recognizes 54,675 individual Affymetrix probe sets, and surveys expression data from 4,142 breast cancer patients (as of 2014). Survival and gene expression data were derived from the GEO (Gene Expression Omnibus), TCGA (The Cancer Genome Atlas), and EGA (European Genomephenome Atlas) databases. In order to ascertain P4HB expression, Affymetrix probe 200654 was selected. Overall Survival in the total population (1402 patients) was determined.

Viability assay.

Proliferation assays were performed as previously described (5). Specifically, the T47Darom cells (aromatase inhibitor sensitive) were plated in 96-well plates at a density of 2×103 cells per well for each cell line and allowed to recover for 24 h. The T47DaromLR cells (AI-resistant) were cultured in the presence of letrozole. For proliferation assays, the cells were treated with DMSO (control) or 30 μM SB205380 in 96-well plates to determine the effects of the various treatments in the absence of letrozole. The resazurin dye (Sigma-Aldrich) was added to each well at 10% of the total volume and measured at 24 h. The SpectraMax® MiniMax® 300 Imaging Cytometer (Molecular Devices, San Jose, CA, USA) was used to measure fluorescence and background wavelengths at 530 nm and 590 nm to determine proliferation. All experiments were performed with n ≥3 and a total of 3 biological replicates were performed. The proliferative activity was calculated as a percent of the vehicle controls as follows:

Antiproliferative activity=[Fluorescence of viable cells (control) − Fluorescence of viable cells (treated)]/Fluorescence of viable cells (control).

Caspase 3/7 apoptosis assay.

The T47Darom and T47DaromLR cells were seeded at a density of 1.5×103 cells in a 96-well plate with appropriate media overnight. Cells were treated the following day with DMSO (control) or 10 μM SB205380. Cells were incubated for 24 h at 37°C with 5% CO2. On the following day, the cells treated with 4 μM CellEvent™ Caspase-3/7 Green Detection Reagent (Invitrogen) and incubated for 30 min at 37°C with 5% CO2. The cells were then fixed with 4% formaldehyde and allowed to incubate for 15 min at room temperature. The SpectraMax®MiniMax® 300 Imaging Cytometer (Molecular Devices) was used to measure fluorescence and background wavelengths at 530 nm and 502 nm, respectively. Cells were imaged on Nikon Ti Eclipse microscope (Minato, Tokyo, Japan) using a FITC filter.

Results

Proteomic profile of estrogen-dependent letrozole resistant breast cancer cells is associated with a unique signature.

To examine the global changes in protein expression as cells transition from AI-sensitive to AI-resistant while maintaining ER expression, a gel-free proteomic approach combining tandem mass tag (TMT) labeling, two-dimensional HPLC, and high-resolution mass spectrometry was utilized. The identities of over 1900 proteins with quantitative abundance ratios were present in both the letrozole sensitive (T47Darom) and the letrozole resistant (T47DaromLR) breast cancer cells. Abundance ratios ≥1.25 were defined as high while those ≤0.8 were defined as low. Of the quantified proteins, a total of 127 were significantly high (p<0.05) as indicated by Table I, while 111 were significantly low (p<0.05) as shown by Table II. The top three up-regulated proteins were mitogen-activated kinase kinase kinase 6 (M3K6), protein disulfide isomerase (P4HB), and midasin (MDN1), and they were increased by 3.204-fold (p<0.01), 3.195-fold (p<0.01), and 2.759-fold (p<0.001), respectively. These proteins are indicated in bold. Among the downregulated proteins, Peptidyl-prolyl cis-trans isomerase (FKBP4) was decreased by 0.484-fold (p<0.001) which is also indicated in bold.

Table I.

Up-regulated proteins in letrozole resistant T47DaromLR breast cancer cells. Proteomic analyses were performed as described in the Methods section and the ratio of the fold change in T47DaromLR/T47Darom cells is shown. Text in bold indicates the proteins exhibiting the greatest fold change.

Accession Description # AAs MW [kDa] Fold change t-test (p-Value)
O95382 Mitogen-activated protein kinase 6 OS=Homo sapiens GN=MAP3K6 PE=1 SV=3 - [M3K6_HUMAN] 1,288 142.506 3.2037514 p<0.01
H0Y3Z3 Protein disulfide-isomerase (Fragment) OS=Homo sapiens GN=P4HB PE=1 SV=1 - [H0Y3Z3_HUMAN] 274 31.5234 3.1950322 p<0.01
Q9NU22 Midasin OS=Homo sapiens GN=MDN1 PE=1 SV=2 - [MDN1_HUMAN] 5,596 632.42 2.7587754 p<0.001
R4GN98 Protein S100 (Fragment) OS=Homo sapiens GN=S100A6 PE=1 SV=1 - [R4GN98_HUMAN] 85 9.67506 2.6447744 p<0.01
P60903 Protein S100-A10 OS=Homo sapiens GN=S100A10 PE=1 SV=2 - [S10AA_HUMAN] 97 11.1955 2.5544666 p<0.001
K7EQI0 Paragranulin (Fragment) OS=Homo sapiens GN=GRN PE=4 SV=1 - [K7EQI0_HUMAN] 110 11.8242 2.4237794 p<0.01
P04083 Annexin A1 OS=Homo sapiens GN=ANXA1 PE=1 SV=2 - [ANXA1_HUMAN] 346 38.69 2.1587977 p<0.001
E5RG29 Aspartyl/asparaginyl beta-hydroxylase (Fragment) OS=Homo sapiens GN=ASPH PE=4 SV=1 - [E5RG29_HUMAN] 198 22.2265 2.1382553 p<0.01
H0YA55 Serum albumin (Fragment) OS=Homo sapiens GN=ALB PE=1 SV=1 - [H0YA55_HUMAN] 454 51.5358 2.129391 p<0.001
A0A087WYQ7 Kynureninase (Fragment) OS=Homo sapiens GN=KYNU PE=1 SV=1 - [A0A087WY Q7_HUMAN] 157 17.3711 2.0269261 p<0.05
P07476 Involucrin OS=Homo sapiens GN=IVL PE=1 SV=2 - [INV0_HUMAN] 585 68.4372 1.9934225 p<0.01
F5H7F6 Microsomal glutathione S-transferase 1 (Fragment)
OS=Homo sapiens GN=MGST1 PE=1 SV=1 - [F5H7F6_HUMAN]		 77
2,997		 8.88048
335.717		 1.8163795
1.8045951		 p<0.001
p<0.01
Q9P2D1 Chromodomain-helicase-DNA-binding protein 7 OS=Homo sapiens GN=CHD7 PE=1 SV=3 - [CHD7_HUMAN]
076070 Gamma-synuclein OS=Homo sapiens GN=SNCG PE=1 SV=2 - [SYUG_HUMAN] 127 13.3228 1.7970365 p<0.001
P07355 Annexin A2 OS=Homo sapiens GN=ANXA2 PE=1 SV=2 - [ANXA2_HUMAN] 339 38.5798 1.7904175 p<0.001
E9PKU7 Neutral alpha-glucosidase AB OS=Homo sapiens GN=GANAB PE=1 SV=1 - [E9PKU7_HUMAN] 852 96.4831 1.7543555 p<0.001
P31949 Protein S100-A11 OS=Homo sapiens GN=S100A11 PE=1 SV=2 - [S 10AB_HUMAN] 105 11.7328 1.7525625 p<0.01
P08729 Keratin, type II cytoskeletal 7 OS=Homo sapiens GN=KRT7 PE=1 SV=5 - [K2C7_HUMAN] 469 51.3543 1.7525381 p<0.001
Q14019 Coactosin-like protein OS=Homo sapiens GN=COTL1 PE=1 SV=3 - [C0TL1_HUMAN] 142 15.935 1.7007204 p<0.001
A0A087WUZ6 InaD-like protein OS=Homo sapiens GN=INADL PE=1 SV=1 - [A0A087WU Z6_HUMAN] 1,182 130 1.6896547 p<0.001
P50897 Palmitoyl-protein thioesterase 1 OS=Homo sapiens GN=PPT1 PE=1 SV=1 - [PPT1_HUMAN] 306 34.1713 1.6864107 p<0.001
R4GN18 Membrane cofactor protein (Fragment) OS=Homo sapiens GN=CD46 PE=4 SV=1 - [R4GN18_HUMAN] 78 8.4965 1.636669 p<0.001
P02647 Apolipoprotein A-I OS=Homo sapiens GN=AP0A1 PE=1 SV=1 - [APOA1 _HUMAN] 267 30.7589 1.624178 p<0.001
Q8WWA1 Transmembrane protein 40 OS=Homo sapiens GN=TMEM40 PE=1 SV=2 - [TMM40_HUMAN] 233 25.4793 1.6170389 p<0.001
P09467 Fructose-1,6-bisphosphatase 1 OS=Homo sapiens GN=FBP1 PE=1 SV=5 - [F16P1 _HUMAN] 338 36.8188 1.6147259 p<0.001
Q15847 Adipogenesis regulatory factor OS=Homo sapiens GN=ADIRF PE=1 SV=1 - [ADIRF_HUMAN] 76 7.84997 1.6089368 p<0.01
O76062 Delta(14)-sterol reductase OS=Homo sapiens GN=TM7SF2 PE=2 SV=3 - [ERG24_HUMAN] 418 46.3755 1.608441 p<0.001
P05026 Sodium/potassium-transporting ATPase subunit beta-1 OS=Homo sapiens GN=ATP1B1 PE=1 SV=1 - [AT1B 1_HUMAN] 303 35.0389 1.6033027 p<0.001
P12532 Creatine kinase U-type, mitochondrial OS=Homo sapiens GN=CKMT1A PE=1 SV=1 - [KCRU_HUMAN] 417 47.0072 1.595103 p<0.001
P02533 Keratin, type I cytoskeletal 14 OS=Homo sapiens GN=KRT14 PE=1 SV=4 - [K1C14_HUMAN] 472 51.5294 1.5900658 p<0.01
F5H2R5 Rho GDP-dissociation inhibitor 2 (Fragment) OS=Homo sapiens GN=ARHGDIB PE=1 SV=1 - [F5H2R5_HUMAN] 88 9.78591 1.5776326 p<0.001
P30038 Delta-1-pyrroline-5-carboxylate dehydrogenase, mitochondrial OS=Homo sapiens GN=ALDH4A1 PE=1 SV=3 - [AL4A1_HUMAN] 563 61.6806 1.5699616 p<0.001
J3KQ45 Trans-Golgi network integral membrane protein 2 OS=Homo sapiens GN=TGOLN2 PE=1 SV=1 - [J3KQ45_HUMAN] 454 47.8517 1.5653502 p<0.001
Q96C23 Aldose 1-epimerase OS=Homo sapiens GN=GALM PE=1 SV=1 - [GALM_HUMAN] 342 37.7421 1.5599502 p<0.001
O15173 Membrane-associated progesterone receptor component 2 OS=Homo sapiens GN=PGRMC2 PE=1 SV=1 - [PGRC2_HUMAN] 223 23.8037 1.5508404 p<0.001
P14406 Cytochrome c oxidase subunit 7A2, mitochondrial OS=Homo sapiens GN=COX7A2 PE=1 SV=1 - [CX7A2_HUMAN] 83 9.39011 1.5499904 p<0.001
F5GYS3 Bcl2-associated agonist of cell death OS=Homo sapiens GN=BAD PE=1 SV=1 - [F5GYS3_HUMAN] 157 16.6392 1.5392774 p<0.01
Q13011 Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase, mitochondrial OS=Homo sapiens GN=ECH1 PE=1 SV=2 - [ECH1 _HUMAN] 328 35.7934 1.5385737 p<0.01
P05091 Aldehyde dehydrogenase, mitochondrial OS=Homo sapiens GN=ALDH2 PE=1 SV=2 - [ALDH2_HUMAN] 517 56.3456 1.5385587 p<0.001
Q5JQF8 Polyadenylate-binding protein 1-like 2 OS=Homo sapiens GN=PAB PC 1L2A PE=2 SV=1 - [PAP1M_HUMAN] 200 22.7846 1.5341699 p<0.001
I3L0Y5 ATP synthase F(0) complex subunit C1, mitochondrial (Fragment) OS=Homo sapiens GN=ATP5G1 PE=3 SV=1 - [I3L0Y 5_HUMAN] 98 10.0322 1.5333557 p<0.05
K7EKD8 Calpain small subunit 1 OS=Homo sapiens GN=CAPNS1 PE=1 SV=1 - [K7EKD8_HUMAN] 238 24.786 1.5273755 p<0.001
P25815 Protein S100-P OS=Homo sapiens GN=S100P PE=1 SV=2 - [S100P_HUMAN] 95 10.3932 1.4987172 p<0.01
E9PB18 UPF0577 protein KIAA1324 (Fragment) OS=Homo sapiens GN=KIAA1324 PE=4 SV=1 - [E9PB 18_HUMAN] 901 99.3015 1.4958927 p<0.001
P07237 Protein disulfide-isomerase OS=Homo sapiens GN=P4HB PE=1 SV=3 - [PDIA1_HUMAN] 508 57.0807 1.4837204 p<0.001
Q9UHB6 LIM domain and actin-binding protein 1 OS=Homo sapiens GN=LIMA1 PE=1 SV=1 - [LIMA 1 _HUMAN] 759 85.1734 1.4787853 p<0.001
P18463 HLA class I histocompatibility antigen, B-37 alpha chain OS=Homo sapiens GN=HLA-B PE=1 SV=1 - [1B37_HUMAN] 362 40.4311 1.4620279 p<0.001
A0A087WT15 Putative bifunctional UDP-N-acetylglucosamine transferase and deubiquitinase ALG13 OS=Homo sapiens GN=ALG13 PE=4 SV=1 - [ A0A087WT15_HUMAN] 61 6.87346 1.4589875 p<0.01
P40121 Macrophage-capping protein OS=Homo sapiens GN=CAPG PE=1 SV=2 - [CAPG_HUMAN] 348 38.4745 1.4573444 p<0.001
Q9Y2Q3 Glutathione S-transferase kappa 1 OS=Homo sapiens GN=GSTK1 PE=1 SV=3 - [GSTK1_HUMAN] 226 25.4803 1.4570236 p<0.001
P04062 Glucosylceramidase OS=Homo sapiens GN=GBA PE=1 SV=3 - [GLCM_HUMAN] 536 59.6782 1.4367309 p<0.001
Q9UBM7 7-dehydrocholesterol reductase OS=Homo sapiens GN=DHCR7 PE=1 SV=1 - [DHCR7_HUMAN] 475 54.4538 1.4332229 p<0.001
Q9Y6N5 Sulfide:quinone oxidoreductase, mitochondrial OS=Homo sapiens GN=SQRDL PE=1 SV=1 - [SQRD_HUMAN] 450 49.9289 1.4319068 p<0.001
P00367 Glutamate dehydrogenase 1, mitochondrial OS=Homo sapiens GN=GLUD1 PE=1 SV=2 - [DHE3_HUMAN] 558 61.3592 1.4305508 p<0.001
O95716 Ras-related protein Rab-3D OS=Homo sapiens GN=RAB3D PE=1 SV=1 - [RAB3D_HUMAN] 219 24.2518 1.4262913 p<0.001
Q24JP5 Transmembrane protein 132A OS=Homo sapiens GN=TMEM132A PE=2 SV=1 - [T13 2A_HUMAN] 1,023 110.041 1.4186958 p<0.001
D6RA82 Annexin OS=Homo sapiens GN=ANXA3 PE=1 SV=1 - [D6RA82_HUMAN] 284 32.0985 1.4022738 p<0.001
P53007 Tricarboxylate transport protein, mitochondrial OS=Homo sapiens GN=SLC25A1 PE=1 SV=2 - [TXTP_HUMAN] 311 33.991 1.3987563 p<0.001
Q8TE77 Protein phosphatase Slingshot homolog 3 OS=Homo sapiens GN=SSH3 PE=1 SV=2 - [SSH3_HUMAN] 659 72.9505 1.3976351 p<0.001
Q10589 Bone marrow stromal antigen 2 OS=Homo sapiens GN=BST2 PE=1 SV=1 - [BST2_HUMAN] 180 19.7563 1.3894274 p<0.001
F8VV56 CD63 antigen OS=Homo sapiens GN=CD63 PE=1 SV=1 - [F8VV56_HUMAN] 145 16.0099 1.3848263 _p<0.05
Q14165 Malectin OS=Homo sapiens GN=MLEC PE=1 SV=1 - [MLEC_HUMAN] 292 32.2136 1.3751279 p<0.001
A3KMH1 von Willebrand factor A domain-containing protein 8 OS=Homo sapiens GN=VWA8 PE=1 SV=2 - [VWA8_HUMAN] 1,905 214.689 1.3749854 p<0.01
P10606 Cytochrome c oxidase subunit 5B, mitochondrial OS=Homo sapiens GN=COX5B PE=1 SV=2 - [COX5B_HUMAN] 129 13.6869 1.3698091 p<0.01
A0A087WVJ0 Mucin-1 (Fragment) OS=Homo sapiens GN=MUC1 PE=4 SV=1 - [A0A087WVJ0_HUMAN] 228 25.5498 1.3697826 p<0.001
Q9Y3Q3 Transmembrane emp24 domain-containing protein 3 OS=Homo sapiens GN=TMED3 PE=1 SV=1 - [TMED3_HUMAN] 217 24.7613 1.3645039 p<0.01
P07686 Beta-hexosaminidase subunit beta OS=Homo sapiens GN=HEXB PE=1 SV=3 - [HEXB_HUMAN] 556 63.0712 1.3635416 p<0.001
O95816 BAG family molecular chaperone regulator 2 OS=Homo sapiens GN=BAG2 PE=1 SV=1 - [BAG2_HUMAN] 211 23.7572 1.3633407 p<0.01
P09525 Annexin A4 OS=Homo sapiens GN=ANXA4 PE=1 SV=4 - [ANXA4_HUMAN] 319 35.8601 1.3605119 p<0.001
Q9Y394 Dehydrogenase/reductase SDR family member 7 OS=Homo sapiens GN=DHRS7 PE=1 SV=1 - [DHRS7_HUMAN] 339 38.2741 1.3556808 p<0.001
P58107 Epiplakin OS=Homo sapiens GN=EPPK1 PE=1 SV=2 - [EPIPL_HUMAN] 5,090 555.279 1.3520266 p<0.001
P00491 Purine nucleoside phosphorylase OS=Homo sapiens GN=PNP PE=1 SV=2 - [PNPH_HUMAN] 289 32.0972 1.3504636 p<0.001
O00625 Pirin OS=Homo sapiens GN=PIR PE=1 SV=1 - [PIR_HUMAN] 290 32.0933 1.3476629 p<0.01
Q9BVC6 Transmembrane protein 109 OS=Homo sapiens GN=TMEM109 PE=1 SV=1 - [TM109_HUMAN] 243 26.1936 1.343218 p<0.001
Q9NR28 Diablo homolog, mitochondrial OS=Homo sapiens GN=DIABLO PE=1 SV=1 - [DBLOH_HUMAN] 239 27.1137 1.3408889 p<0.01
Q86UP2 Kinectin OS=Homo sapiens GN=KTN1 PE=1 SV=1 - [KTN1_HUMAN] 1,357 156.179 1.3392072 p<0.001
P04181 Ornithine aminotransferase, mitochondrial OS=Homo sapiens GN=OAT PE=1 SV=1 - [OAT_HUMAN] 439 48.5042 1.3330075 p<0.001
O15270 Serine palmitoyltransferase 2 OS=Homo sapiens GN=SPTLC2 PE=1 SV=1 - [SPTC2_HUMAN] 562 62.8839 1.3326487 p<0.05
E9PF16 Acyl-CoA synthetase family member 2, mitochondrial OS=Homo sapiens GN=ACSF2 PE=4 SV=1 - [E9PF16_HUMAN] 572 63.6114 1.3315351 p<0.01
Q14956 Transmembrane glycoprotein NMB OS=Homo sapiens GN=GPNMB PE=1 SV=2 - [GPNMB_HUMAN] 572 63.8819 1.3315248 p<0.01
O75531 Barrier-to-autointegration factor OS=Homo sapiens GN=BANF1 PE=1 SV=1 - [BAF_HUMAN] 89 10.052 1.3302003 p<0.01
Q9H0U4 Ras-related protein Rab-1B OS=Homo sapiens GN=RAB1B PE=1 SV=1 - [RAB1 B_HUMAN] 201 22.1572 1.3258098 p<0.001
P05783 Keratin, type I cytoskeletal 18 OS=Homo sapiens GN=KRT18 PE=1 SV=2 - [K1C18_HUMAN] 430 48.0285 1.3239039 p<0.001
Q9UDW1 Cytochrome b-c1 complex subunit 9 OS=Homo sapiens GN=UQCR10 PE=1 SV=3 - [QCR9_HUMAN] 63 7.30383 1.3232184 p<0.001
H3BM67 Nucleolar protein 3 (Fragment) OS=Homo sapiens GN=NOL3 PE=4 SV=3 - [H3BM67_HUMAN] 139 15.0125 1.3207562 p<0.01
P50454 Serpin H1 OS=Homo sapiens GN=SERPINH1 PE=1 SV=2 - [SERPH_HUMAN] 418 46.4112 1.3189995 p<0.001
P15529 Membrane cofactor protein OS=Homo sapiens GN=CD46 PE=1 SV=3 - [MCP_HUMAN] 392 43.7187 1.3184128 p<0.01
J3KT68 Transmembrane protein 97 OS=Homo sapiens GN=TMEM97 PE=1 SV=1 - [J3KT68_HUMAN] 92 10.9688 1.3162762 p<0.01
P35232 Prohibitin OS=Homo sapiens GN=PHB PE=1 SV=1 - [PHB_HUMAN] 272 29.7859 1.3155472 p<0.001
P29373 Cellular retinoic acid-binding protein 2 OS=Homo sapiens GN=CRABP2 PE=1 SV=2 - [RABP2_HUMAN] 138 15.683 1.3147 p<0.001
Q9BR76 Coronin-1B OS=Homo sapiens GN=CORO1B PE=1 SV=1 - [COR1B_HUMAN] 489 54.2003 1.3079348 p<0.001
P61604 10 kDa heat shock protein, mitochondrial OS=Homo sapiens GN=HSPE1 PE=1 SV=2 - [CH10_HUMAN] 102 10.9249 1.3066874 p<0.01
P14314 Glucosidase 2 subunit beta OS=Homo sapiens GN=PRKCSH PE=1 SV=2 - [GLU2B_HUMAN] 528 59.3878 1.3050784 p<0.001
H0YDJ4 Ninein (Fragment) OS=Homo sapiens GN=NIN PE=1 SV=1 - [H0YDJ4_HUMAN] 1,537 179.439 1.3034976 p<0.01
E7ERY9 Calcium-transporting ATPase OS=Homo sapiens GN=ATP2B1 PE=1 SV=2 - [E7ERY 9_HUMAN] 963 106.789 1.30286 p<0.001
P26885 Peptidyl-prolyl cis-trans isomerase FKBP2 OS=Homo sapiens GN=FKBP2 PE=1 SV=2 - [FKBP2_HUMAN] 142 15.6393 1.3022129 p<0.001
Q9BTV4 Transmembrane protein 43 OS=Homo sapiens GN=TMEM43 PE=1 SV=1 - [TMM43_HUMAN] 400 44.8472 1.3012834 p<0.001
Q15075 Early endosome antigen 1 OS=Homo sapiens GN=EEA1 PE=1 SV=2 - [EEA1 _HUMAN] 1,411 162.367 1.2999296 p<0.001
P04004 Vitronectin OS=Homo sapiens GN=VTN PE=1 SV=1 - [VTNC_HUMAN] 478 54.2712 1.2952339 p<0.001
Q6P996 Pyridoxal-dependent decarboxylase domain-containing protein 1 OS=Homo sapiens GN=PDXDC1 PE=1 SV=2 - [PDXD1_HUMAN] 788 86.6523 1.2938765 p<0.05
P04179 Superoxide dismutase [Mn], mitochondrial OS=Homo sapiens GN=SOD2 PE=1 SV=2 - [SODM_HUMAN] 222 24.7066 1.293093 p<0.001
M0QYZ0 Heterogeneous nuclear ribonucleoprotein U-like protein 1 (Fragment) OS=Homo sapiens GN=HNRNPUL1 PE=1 SV=1 - [M0QYZ0_HUMAN] 371 42.2697 1.2927079 p<0.001
Q9BZQ8 Protein Niban OS=Homo sapiens GN=FAM129A PE=1 SV=1 - [NIBAN_HUMAN] 928 103.07 1.2916002 p<0.001
P00390 Glutathione reductase, mitochondrial OS=Homo sapiens GN=GSR PE=1 SV=2 - [GSHR_HUMAN] 522 56.221 1.290853 p<0.001
K7EM09 Transmembrane protein 205 (Fragment) OS=Homo sapiens GN=TMEM205 PE=1 SV=1 - [K7EM09_HUMAN] 120 13.3807 1.2885968 p<0.001
I3L3E4 Charged multivesicular body protein 6 (Fragment) OS=Homo sapiens GN=CHMP6 PE=1 SV=1 - [I3L3E4_HUMAN] 113 12.9314 1.2840595 p<0.05
Q9BX68 Histidine triad nucleotide-binding protein 2, mitochondrial OS=Homo sapiens GN=HINT2 PE=1 SV=1 - [HINT2_HUMAN] 163 17.1512 1.2837525 p<0.001
P45973 Chromobox protein homolog 5 OS=Homo sapiens GN=CBX5 PE=1 SV=1 - [CBX5_HUMAN] 191 22.2111 1.281572 p<0.001
P05787 Keratin, type II cytoskeletal 8 OS=Homo sapiens GN=KRT8 PE=1 SV=7 - [K2C8_HUMAN] 483 53.6711 1.2780591 p<0.001
H0Y339 E3 ubiquitin-protein ligase RFWD2 (Fragment) OS=Homo sapiens GN=RFWD2 PE=4 SV=1 - [H0Y339_HUMAN] 566 64.4877 1.2775849 p<0.01
P02787 Serotransferrin OS=Homo sapiens GN=TF PE=1 SV=3 - [TRFE_HUMAN] 698 77.0136 1.2759193 p<0.01
A8MT65 Zinc finger protein 891 OS=Homo sapiens GN=ZNF891 PE=2 SV=2 - [ZN891_HUMAN] 544 63.5502 1.2734141 p<0.01
O75874 Isocitrate dehydrogenase [NADP] cytoplasmic OS=Homo sapiens GN=IDH1 PE=1 SV=2 - [IDHC_HUMAN] 414 46.6295 1.2724354 p<0.001
P12236 ADP/ATP translocase 3 OS=Homo sapiens GN=SLC25A6 PE=1 SV=4 - [ADT3_HUMAN] 298 32.8452 1.2713881 p<0.01
O75131 Copine-3 OS=Homo sapiens GN=CPNE3 PE=1 SV=1 - [CPNE3_HUMAN] 537 60.0921 1.2713204 p<0.001
F8VVM2 Phosphate carrier protein, mitochondrial OS=Homo sapiens GN=SLC25A3 PE=1 SV=1 - [F8VVM2_HUMAN] 324 36.1376 1.2702845 p<0.001
Q2TAY7 WD40 repeat-containing protein SMU1 OS=Homo sapiens GN=SMU1 PE=1 SV=2 - [SMU1_HUMAN] 513 57.5072 1.2690306 p<0.001
Q15293 Reticulocalbin-1 OS=Homo sapiens GN=RCN1 PE=1 SV=1 - [RCN1_HUMAN] 331 38.8662 1.2635162 p<0.001
E9PM95 Apolipoprotein L2 (Fragment) OS=Homo sapiens GN=APOL2 PE=1 SV=1 - [E9PM95_HUMAN] 128 14.9386 1.2630585 p<0.01
A0A087X208 Agrin OS=Homo sapiens GN=AGRN PE=4 SV=1 - [A0A087X208_HUMAN] 1,930 202.162 1.2620734 p<0.001
A0A087WY61 Nuclear mitotic apparatus protein 1 OS=Homo sapiens GN=NUMA1 PE=1 SV=1 - [A0A087WY61 _HUMAN] 2,099 236.019 1.2614504 p<0.001
Q16698 2,4-dienoyl-CoA reductase, mitochondrial OS=Homo sapiens GN=DECR1 PE=1 SV=1 - [DECR_HUMAN] 335 36.0448 1.2589002 p<0.001
Q14011 Cold-inducible RNA-binding protein OS=Homo sapiens GN=CIRBP PE=1 SV=1 - [CIRBP_HUMAN] 172 18.6367 1.2565207 p<0.001
P14625 Endoplasmin OS=Homo sapiens GN=HSP90B1 PE=1 SV=1 - [ENPL_HUMAN] 803 92.4113 1.2542374 p<0.001
Q96HE7 ERO1-like protein alpha OS=Homo sapiens GN=ERO1L PE=1 SV=2 - [ERO1A_HUMAN] 468 54.3581 1.2516932 p<0.001
P17900 Ganglioside GM2 activator OS=Homo sapiens GN=GM2A PE=1 SV=4 - [SAP3_HUMAN] 193 20.8247 1.2515007 p<0.001
P27824 Calnexin OS=Homo sapiens GN=CANX PE=1 SV=2 - [CALX_HUMAN] 592 67.5259 1.2502035 p<0.001
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Table II.

Down-regulated proteins in letrozole resistant T47DaromLR breast cancer cells. Proteomic analyses were performed as described in the Methods section and the ratio of the fold change in T47DaromLR/T47Darom cells is shown. The text in bold indicates a protein of interest.

Accession Description # AAs MW [kDa] Fold change t-test (p-value)
Q15185 Prostaglandin E synthase 3 OS=Homo sapiens GN=PTGES3 PE=1 SV=1 - [TEBP_HUMAN] 160 18.6854194 0.79904589 p<0.01
Q9NYL9 Tropomodulin-3 OS=Homo sapiens GN=TMOD3 PE=1 SV=1 - [TMOD3_HUMAN] 352 39.5703018 0.79902558 p<0.05
P18669 Phosphoglycerate mutase 1 OS=Homo sapiens GN=PGAM1 PE=1 SV=2 - [PGAM1_HUMAN] 254 28.7858365 0.79865701 p<0.001
P19338 Nucleolin OS=Homo sapiens GN=NCL PE=1 SV=3 - [NUCL_HUMAN] 710 76.5683628 0.79790721 p<0.001
Q12931 Heat shock protein 75 kDa, mitochondrial OS=Homo sapiens GN=TRAP1 PE=1 SV=3 - [TRAP 1 _HUMAN] 704 80.0596949 0.79777903 p<0.001
Q86Y56 HEAT repeat-containing protein 2 OS=Homo sapiens GN=HEATR2 PE=1 SV=4 - [HEAT2_HUMAN] 855 93.4620686 0.79675307 p<0.001
K7EJE8 Lon protease homolog, mitochondrial OS=Homo sapiens GN=LONP1 PE=1 SV=1 - [K7EJE8_HUMAN] 829 93.2379667 0.79656905 p<0.001
Q8IZ52 Chondroitin sulfate synthase 2 OS=Homo sapiens GN=CHPF PE=1 SV=2 - [CHSS2_HUMAN] 775 85.4140471 0.79611883 p<0.01
P31939 Bifunctional purine biosynthesis protein PURH OS=Homo sapiens GN=ATIC PE=1 SV=3 - [PUR9_HUMAN] 592 64.5753451 0.79607097 p<0.001
O15371 Eukaryotic translation initiation factor 3 subunit D OS=Homo sapiens GN=EIF3D PE=1 SV=1 - [EIF3 D_HUMAN] 548 63.9324345 0.79584502 p<0.001
P12004 Proliferating cell nuclear antigen OS=Homo sapiens GN=PCNA PE=1 SV=1 - [PCNA_HUMAN] 261 28.7502945 0.79483268 p<0.001
P68371 Tubulin beta-4B chain OS=Homo sapiens GN=TUBB4B PE=1 SV=1 - [TBB4B_HUMAN] 445 49.7990043 0.79464307 p<0.01
V9GYX7 Actin-related protein 10 (Fragment) OS=Homo sapiens GN=ACTR10 PE=1 SV=1 - [V9GYX7_HUMAN] 302 33.2901748 0.79372567 p<0.05
P62258 14-3-3 protein epsilon OS=Homo sapiens GN=YWHAE PE=1 SV=1 - [1433E_HUMAN] 255 29.1554174 0.7932199 p<0.001
Q9NZL9 Methionine adenosyltransferase 2 subunit beta OS=Homo sapiens GN=MAT2B PE=1 SV=1 - [MAT2B_HUMAN] 334 37.5281776 0.7926943 p<0.001
P13995 Bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase, mitochondrial OS=Homo sapiens GN=MTHFD2 PE=1 SV=2 - [MTDC_HUMAN] 350 37.8711975 0.79227913 p<0.01
P62826 GTP-binding nuclear protein Ran OS=Homo sapiens GN=RAN PE=1 SV=3 - [RAN_HUMAN] 216 24.4076189 0.791804 p<0.001
P50990 T-complex protein 1 subunit theta OS=Homo sapiens GN=CCT8 PE=1 SV=4 - [TCPQ_HUMAN] 548 59.582509 0.78997166 p<0.001
M0R210 40S ribosomal protein S16 OS=Homo sapiens GN=RPS16 PE=1 SV=1 - [M0R210_HUMAN] 129 14.4099586 0.78837739 p<0.001
Q9NPH2 Inositol-3-phosphate synthase 1 OS=Homo sapiens GN=ISYNA1 PE=1 SV=1 - [INO1_HUMAN] 558 61.029104 0.78806023 p<0.01
Q32Q12 Nucleoside diphosphate kinase OS=Homo sapiens GN=NME 1-NME2 PE=1 SV=1 - [Q32Q12_HUMAN] 292 32.6208685 0.78792838 p<0.001
P40937 Replication factor C subunit 5 OS=Homo sapiens GN=RFC5 PE=1 SV=1 - [RFC5_HUMAN] 340 38.4721873 0.78511694 p<0.01
D6RFM5 Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial OS=Homo sapiens GN=SDHA PE=1 SV=1 - [D6RFM5_HUMAN] 583 63.5266889 0.78479348 p<0.001
H7C3S9 COP9 signalosome complex subunit 8 (Fragment) OS=Homo sapiens GN=COPS8 PE=1 SV=1 - [H7C3S9_HUMAN] 83 9.03868894 0.78280605 p<0.001
Q99536 Synaptic vesicle membrane protein VAT-1 homolog OS=Homo sapiens GN=VAT1 PE=1 SV=2 - [VAT1_HUMAN] 393 41.8934194 0.78202386 p<0.001
Q9BUF5 Tubulin beta-6 chain OS=Homo sapiens GN=TUBB6 PE=1 SV=1 - [TBB6_HUMAN] 446 49.8250022 0.78081583 p<0.05
Q9Y2Z0 Suppressor of G2 allele of SKP1 homolog OS=Homo sapiens GN=SUGT1 PE=1 SV=3 - [SUGT1_HUMAN] 365 40.998492 0.78075718 p<0.05
E7EUH7 Pseudouridylate synthase 7 homolog OS=Homo sapiens GN=PUS7 PE=1 SV=1 - [E7EUH7_HUMAN] 448 50.5206902 0.78060885 p<0.05
Q13907 Isopentenyl-diphosphate Delta-isomerase 1 OS=Homo sapiens GN=IDI1 PE=1 SV=2 - [IDI1_HUMAN] 227 26.3024914 0.7785156 p<0.001
F8VXD2 Cyclin-dependent kinase 4 (Fragment) OS=Homo sapiens GN=CDK4 PE=1 SV=1 - [F8VXD2_HUMAN] 132 14.8906691 0.77756677 p<0.05
P08559 Pyruvate dehydrogenase E1 component subunit alpha, somatic form, mitochondrial OS=Homo sapiens GN=PDHA1 PE=1 SV=3 - [ODPA_HUMAN] 390 43.2676109 0.77727405 p<0.001
O00762 Ubiquitin-conjugating enzyme E2 C OS=Homo sapiens GN=UBE2C PE=1 SV=1 - [UBE2C_HUMAN] 179 19.6399202 0.77693257 p<0.001
P43490 Nicotinamide phosphoribosyltransferase OS=Homo sapiens GN=NAMPT PE=1 SV=1 - [NAMPT_HUMAN] 491 55.4865965 0.77652882 p<0.001
D6RDM7 Ubiquitin-conjugating enzyme E2 K (Fragment) OS=Homo sapiens GN=UBE2K PE=1 SV=1 - [D6RDM7_HUMAN] 123 14.0261903 0.77622448 p<0.001
E5RFP0 NudC domain-containing protein 2 OS=Homo sapiens GN=NUDCD2 PE=1 SV=1 - [E5RFP0_HUMAN] 132 14.7361652 0.77511218 p<0.01
Q04917 14-3-3 protein eta OS=Homo sapiens GN=YWHAH PE=1 SV=4 - [1433F_HUMAN] 246 28.20102 0.77478302 p<0.01
P29144 Tripeptidyl-peptidase 2 OS=Homo sapiens GN=TPP2 PE=1 SV=4 - [TPP2_HUMAN] 1,249 138.262632 0.77415019 p<0.01
P68032 Actin, alpha cardiac muscle 1 OS=Homo sapiens GN=ACTC1 PE=1 SV=1 - [ACTC_HUMAN] 377 41.9918826 0.77329561 p<0.001
Q562R1 Beta-actin-like protein 2 OS=Homo sapiens GN=ACTBL2 PE=1 SV=2 - [ACTBL_HUMAN] 376 41.9759693 0.76932533 p<0.001
Q14914 Prostaglandin reductase 1 OS=Homo sapiens GN=PTGR1 PE=1 SV=2 - [PTGR1_HUMAN] 329 35.8467307 0.76842181 p<0.01
Q05639 Elongation factor 1-alpha 2 OS=Homo sapiens GN=EEF1A2 PE=1 SV=1 - [EF1A2_HUMAN] 463 50.4383096 0.76649072 p<0.001
P06396 Gelsolin OS=Homo sapiens GN=GSN PE=1 SV=1 - [GELS_HUMAN] 782 85.6441934 0.76419352 p<0.001
P11908 Ribose-phosphate pyrophosphokinase 2 OS=Homo sapiens GN=PRPS2 PE=1 SV=2 - [PRPS2_HUMAN] 318 34.7469789 0.76381858 p<0.01
Q58FF6 Putative heat shock protein HSP 90-beta 4 OS=Homo sapiens GN=HSP90AB4P PE=5 SV=1 - [H90B4_HUMAN] 505 58.2275805 0.76277969 p<0.001
H7C3P9 COP9 signalosome complex subunit 3 OS=Homo sapiens GN=COPS3 PE=1 SV=2 - [H7C3P9_HUMAN] 345 39.0105281 0.76178683 p<0.001
P80404 4-aminobutyrate aminotransferase, mitochondrial OS=Homo sapiens GN=ABAT PE=1 SV=3 - [GABT_HUMAN] 500 56.4025903 0.75993164 p<0.001
P17174 Aspartate aminotransferase, cytoplasmic OS=Homo sapiens GN=GOT1 PE=1 SV=3 - [AATC_HUMAN] 413 46.2185309 0.75976861 p<0.001
Q9HB71 Calcyclin-binding protein OS=Homo sapiens GN=CACYBP PE=1 SV=2 - [CYBP_HUMAN] 228 26.1936459 0.75789333 p<0.001
P62328 Thymosin beta-4 OS=Homo sapiens GN=TMSB4X PE=1 SV=2 - [TYB4_HUMAN] 44 5.04951649 0.75606602 p<0.05
E9PNK6 Tumor protein D53 OS=Homo sapiens GN=TPD52L1 PE=1 SV=1 - [E9PNK6_HUMAN] 166 18.6492831 0.75524912 p<0.01
Q96IU4 Alpha/beta hydrolase domain-containing protein 14B OS=Homo sapiens GN=ABHD14B PE=1 SV=1 - [ABHEB_HUMAN] 210 22.3315574 0.75457008 p<0.01
Q9NP72 Ras-related protein Rab-18 OS=Homo sapiens GN=RAB18 PE=1 SV=1 - [RAB18_HUMAN] 206 22.9626389 0.7544287 p<0.001
C9JLU1 DNA-directed RNA polymerases I, II, and III subunit RPABC3 (Fragment) OS=Homo sapiens GN=POLR2H PE=1 SV=3 - [C9JLU 1_HUMAN] 149 16.9853803 0.75427123 p<0.01
Q8N0Y7 Probable phosphoglycerate mutase 4 OS=Homo sapiens GN=PGAM4 PE=2 SV=1 - [PGAM4_HUMAN] 254 28.7587667 0.75337853 p<0.05
E9PLT0 Cold shock domain-containing protein E1 OS=Homo sapiens GN=CSDE1 PE=1 SV=1 - [E9PLT0_HUMAN] 668 74.5367585 0.75176491 p<0.001
P07900 Heat shock protein HSP 90-alpha OS=Homo sapiens GN=HSP90AA1 PE=1 SV=5 - [HS90A_HUMAN] 732 84.6066852 0.75149833 p<0.001
F5H345 Porphobilinogen deaminase OS=Homo sapiens GN=HMBS PE=1 SV=1 - [F5H345_HUMAN] 330 35.7388228 0.75086131 p<0.01
P41227 N-alpha-acetyltransferase 10 OS=Homo sapiens GN=NAA10 PE=1 SV=1 - [NAA10_HUMAN] 235 26.4418527 0.75055952 p<0.001
P35580 Myosin-10 OS=Homo sapiens GN=MYH10 PE=1 SV=3 - [MYH10_HUMAN] 1,976 228.857964 0.75001828 p<0.01
P36507 Dual specificity mitogen-activated protein kinase kinase 2 OS=Homo sapiens GN=MAP2K2 PE=1 SV=1 - [MP2K2_HUMAN] 400 44.396055 0.7484181 p<0.01
Q9UNF1 Melanoma-associated antigen D2 OS=Homo sapiens GN=MAGED2 PE=1 SV=2 - [MAGD2_HUMAN] 606 64.9143628 0.74753167 p<0.001
P61981 14–3-3 protein gamma OS=Homo sapiens GN=YWHAG PE=1 SV=2 - [1433G_HUMAN] 247 28.2849139 0.74714741 p<0.001
Q9Y265 RuvB-like 1 OS=Homo sapiens GN=RUVBL1 PE=1 SV=1 - [RUVB1_HUMAN] 456 50.1963165 0.74346671 p<0.001
Q7L2H7 Eukaryotic translation initiation factor 3 subunit M OS=Homo sapiens GN=EIF3M PE=1 SV=1 - [EIF3M_HUMAN] 374 42.475807 0.74293059 p<0.01
Q9BQE3 Tubulin alpha-1C chain OS=Homo sapiens GN=TUBA1C PE=1 SV=1 - [TBA1C_HUMAN] 449 49.8634645 0.74276939 p<0.001
P13639 Elongation factor 2 OS=Homo sapiens GN=EEF2 PE=1 SV=4 - [EF2_HUMAN] 858 95.2769538 0.73926002 p<0.001
C9JPA8 Golgi to ER traffic protein 4 homolog (Fragment) OS=Homo sapiens GN=GET4 PE=1 SV=2 - [C9JPA8_HUMAN] 148 16.5321422 0.73902176 p<0.001
Q53EL6 Programmed cell death protein 4 OS=Homo sapiens GN=PDCD4 PE=1 SV=2 - [PDCD4_HUMAN] 469 51.7030487 0.73378818 p<0.001
E5RG13 Inositol monophosphatase 1 (Fragment) OS=Homo sapiens GN=IMPA1 PE=1 SV=1 - [E5RG13_HUMAN] 153 17.0476334 0.73217864 p<0.01
O96019 Actin-like protein 6A OS=Homo sapiens GN=ACTL6A PE=1 SV=1 - [ACL6A_HUMAN] 429 47.4303444 0.7298752 p<0.01
O75369 Filamin-B OS=Homo sapiens GN=FLNB PE=1 SV=2 - [FLNB_HUMAN] 2,602 277.990071 0.72651996 p<0.001
P68363 Tubulin alpha-1B chain OS=Homo sapiens GN=TUBA1B PE=1 SV=1 - [TBA1 B_HUMAN] 451 50.1196069 0.72651105 p<0.001
Q9H0C2 ADP/ATP translocase 4 OS=Homo sapiens GN=SLC25A31 PE=2 SV=1 - [ADT4_HUMAN] 315 34.9994373 0.72226342 p<0.01
Q8TC12 Retinol dehydrogenase 11 OS=Homo sapiens GN=RDH11 PE=1 SV=2 - [RDH11_HUMAN] 318 35.3634467 0.72142867 p<0.001
P08238 Heat shock protein HSP 90-beta OS=Homo sapiens GN=HSP90AB1 PE=1 SV=4 - [HS90B_HUMAN] 724 83.2121059 0.71815962 p<0.001
P11177 Pyruvate dehydrogenase E1 component subunit beta, mitochondrial OS=Homo sapiens GN=PDHB PE=1 SV=3 - [ODPB_HUMAN] 359 39.2080346 0.71532722 p<0.001
A0A087WWP8 Round spermatid basic protein 1 OS=Homo sapiens GN=RSBN1 PE=1 SV=1 - [A0A087WWP8_HUMAN] 754 84.922968 0.70870038 p<0.05
P00338 L-lactate dehydrogenase A chain OS=Homo sapiens GN=LDHA PE=1 SV=2 - [LDHA_HUMAN] 332 36.6653627 0.70859232 p<0.001
Q96CP2 FLYWCH family member 2 OS=Homo sapiens GN=FLYWCH2 PE=1 SV=1 - [FWCH2_HUMAN] 140 14.5545451 0.7042261 p<0.05
O00232 26S proteasome non-ATPase regulatory subunit 12 OS=Homo sapiens GN=PSMD12 PE=1 SV=3 - [PSD12_HUMAN] 456 52.8705755 0.70028253 p<0.001
P07437 Tubulin beta chain OS=Homo sapiens GN=TUBB PE=1 SV=2 - [TBB5_HUMAN] 444 49.6389736 0.69867551 p<0.001
Q9HAV0 Guanine nucleotide-binding protein subunit beta-4 OS=Homo sapiens GN=GNB4 PE=1 SV=3 - [GBB4_HUMAN] 340 37.5430236 0.69638699 p<0.001
P62937 Peptidyl-prolyl cis-trans isomerase A OS=Homo sapiens GN=PPIA PE=1 SV=2 - [PPIA_HUMAN] 165 18.0008862 0.69099459 p<0.001
P41250 Glycine--tRNA ligase OS=Homo sapiens GN=GARS PE=1 SV=3 - [SYG_HUMAN] 739 83.1126144 0.68820486 p<0.001
A0A087WW67 Inactive N-acetylated-alpha-linked acidic dipeptidase-like protein 2 OS=Homo sapiens GN=NAALADL2 PE=4 SV=1 - [A0A087WW 67_HUMAN] 783 87.4024178 0.68607324 p<0.01
P52735 Guanine nucleotide exchange factor VAV2 OS=Homo sapiens GN=VAV2 PE=1 SV=2 - [VAV2_HUMAN] 878 101.223761 0.68510642 p<0.001
E9PCZ3 Type II inositol 3,4-bisphosphate 4-phosphatase (Fragment) OS=Homo sapiens GN=INPP4B PE=4 SV=1 - [E9PCZ3_HUMAN] 688 78.1047694 0.67949237 p<0.001
P09382 Galectin-1 OS=Homo sapiens GN=LGALS1 PE=1 SV=2 - [LEG 1 _HUMAN] 135 14.7062001 0.67748938 p<0.001
P48052 Carboxypeptidase A2 OS=Homo sapiens GN=CPA2 PE=1 SV=3 - [CBPA2_HUMAN] 419 47.0006266 0.67601541 p<0.01
K7ERE3 Keratin, type I cytoskeletal 13 OS=Homo sapiens GN=KRT13 PE=1 SV=1 - [K7ERE3_HUMAN] 415 45.232239 0.66812005 p<0.001
B3KUB4 Carbonic anhydrase 12 OS=Homo sapiens GN=CA12 PE=2 SV=1 - [B3KUB4_HUMAN] 283 31.8373497 0.66653472 p<0.001
Q5JVM0 Unconventional myosin-VI (Fragment) OS=Homo sapiens GN=MY06 PE=1 SV=1 - [Q5JVM0_HUMAN] 174 20.5885602 0.66103342 p<0.001
Q13451 Peptidyl-prolyl cis-trans isomerase FKBP5 OS=Homo sapiens GN=FKBP5 PE=1 SV=2 - [FKBP5_HUMAN] 457 51.1795705 0.66044757 p<0.001
P21266 Glutathione S-transferase Mu 3 OS=Homo sapiens GN=GSTM3 PE=1 SV=3 - [GSTM3_HUMAN] 225 26.5421389 0.65763968 p<0.001
F5GWT4 Serine/threonine-protein kinase WNK1 OS=Homo sapiens GN=WNK1 PE=1 SV=1 - [F5GWT4_HUMAN] 2,134 225.364792 0.65376951 p<0.01
Q9BV86 N-terminal Xaa-Pro-Lys N-methyltransferase 1 OS=Homo sapiens GN=NTMT1 PE=1 SV=3 - [NTM1 A_HUMAN] 223 25.3708399 0.65311967 p<0.001
014907 Tax1-binding protein 3 OS=Homo sapiens GN=TAX1BP3 PE=1 SV=2 - [TX1B3_HUMAN] 124 13.7261295 0.62476735 p<0.001
014745 Na(+)/H(+) exchange regulatory cofactor NHE-RF1 OS=Homo sapiens GN=SLC9A3R1 PE=1 SV=4 - [NHRF1 _HUMAN] 358 38.844612 0.6105772 p<0.001
A0A096LPF0 Ras-related protein Rab-7b (Fragment) OS=Homo sapiens GN=RAB7B PE=4 SV=1 - [A0A096LPF0_HUMAN] 91 10.3803502 0.59567283 p<0.001
Q8IVL1 Neuron navigator 2 OS=Homo sapiens GN=NAV2 PE=1 SV=3 - [NAV2_HUMAN] 2,488 268.002547 0.58546377 p<0.001
Q9UKY7 Protein CDV3 homolog OS=Homo sapiens GN=CDV3 PE=1 SV=1 - [CDV 3_HUMAN] 258 27.3183615 0.56625057 p<0.001
B4DEY6 LIM and cysteine-rich domains protein 1 OS=Homo sapiens GN=LMCD1 PE=2 SV=1 - [B4DEY 6_HUMAN] 253 28.4947789 0.56611995 p<0.01
P49006 MARCKS-related protein OS=Homo sapiens GN=MARCKSL1 PE=1 SV=2 - [MRP_HUMAN] 195 19.5172325 0.56466249 p<0.01
P36405 ADP-ribosylation factor-like protein 3 OS=Homo sapiens GN=ARL3 PE=1 SV=2 - [ARL3_HUMAN] 182 20.44276 0.56320138 p<0.001
Q9Y2T7 Y-box-binding protein 2 OS=Homo sapiens GN=YBX2 PE=1 SV=2 - [YB0X2_HUMAN] 364 38.4947655 0.55489064 p<0.01
H0YE40 CD44 antigen (Fragment) OS=Homo sapiens GN=CD44 PE=1 SV=1 - [H0YE40_HUMAN] 82 9.09947282 0.51939027 p<0.001
P06401 Progesterone receptor OS=Homo sapiens GN=PGR PE=1 SV=4 - [PRGR_HUMAN] 933 98.9181716 0.5090052 p<0.001
Q02790 Peptidyl-prolyl cis-trans isomerase FKBP4 OS=Homo sapiens GN=FKBP4 PE=1 SV=3 - [FKBP4_HUMAN] 459 51.7720738 0.48439102 p<0.001
P08185 Corticosteroid-binding globulin OS=Homo sapiens GN=SERPINA6 PE=1 SV=1 - [CBG_HUMAN] 405 45.1119093 0.39363177 p<0.001
Q9H993 UPF0364 protein C6orf211 OS=Homo sapiens GN=C6orf211 PE=1 SV=1 - [CF211_HUMAN] 441 51.1398629 0.37407832 p<0.001
P02795 Metallothionein-2 OS=Homo sapiens GN=MT2A PE=1 SV=1 - [MT2_HUMAN] 61 6.0371964 0.33853248 p<0.001
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High MAP3K6 expression is associated with decreased relapse-free survival.

Since MAP3K6 was significantly increased, we sought to examine the clinical relevance on patient survival. To predict whether increased MAP3K6 expression levels were associated with differences in survival, KM Plotter was used to interrogate publicly available microarray repositories of breast cancer patients. Based on these parameters, high MAP3K6 expression was found to be associated with a decrease in relapse-free survival (p=0.0019) with a hazard ratio of 0.84 (0.75–0.84) (Figure 1).

Figure 1.

Figure 1.

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High MAP3K6 expression is associated with decreased relapse-free survival in breast cancer patients. Using Kaplan–Meier Plotter (KM Plotter), publicly available microarray repositories for breast cancer were interrogated to determine whether MAP3K6 expression was associated with different survival rates among breast cancer patients. Hazard ratio (HR) and Log-rank p-values are shown. Low expression (below median) is noted in black, and the high expression (above median) is noted in red.

The MAPK signaling cascade is activated as cells acquire resistance to letrozole.

Since M3K6 exhibited the highest increase in expression between the two cell lines, we were interested in determining whether this alteration was associated with activation of the MAPK signaling cascades as a result of ER+ letrozole resistance. To study multiple proteins within this pathway and other receptor tyrosine kinase signaling cascades, the MAPK and RTK phospho-antibody arrays were utilized to measure the activation of key signaling components between the T47Darom and the T47DaromLR cells. We chose both arrays because a previous finding from our group demonstrated that HER2 expression was increased in the T47DaromLR cells (6). Although total HER2 expression was previously shown to be increased, activated ErbB2 (HER2) levels were decreased by 1.40-fold (p<0.05), suggesting this may not be a critical driver associated with ER+ AI resistance (Figure 2). We also observed increased expression of IGF1R which was previously found up-regulated in the LTLT-Ca cells (7).

Figure 2.

Figure 2.

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Hormone dependent letrozole resistance induces global down-regulation of activated receptor tyrosine kinases. Graph depict normalized mean pixel density of protein levels in lysates prepared from the T47Darom and T47DaromLR cells using the Human RTK Antibody Array Membrane where 71 different anti-RTK antibodies were spotted in duplicated, including 4 positive and 3 negative controls and 1 blank. Array signals were analyzed using Image Lab (BioRad) software. Values from duplicate spots were averaged and plotted. Results are expressed as the mean unit±SD (****p<0.0001, ***p<0.001, **p<0.01, *p<0.05).

When signaling components associated with the MAPK pathway were assessed, several phosphorylated proteins of interest were significantly increased including MKK6 (1.20-fold), p38 (1.41-fold), p70S6K (1.74-fold), phospho-Ribosomal S6 Kinase 1 (1.71-fold) and phospho-Ribosomal S6 Kinase 2 (4.74-fold) (Figure 3). This was highly relevant as this pathway is associated with growth, differentiation, apoptosis, and survival. Since there was not a significant change in Akt and mTOR expression, it is unlikely that the PI3K/Akt/mTOR pathway plays a major role in ER+ acquired AI resistance. Additionally, since both MEK and ERK1 were significantly decreased by 1.43- and 1.08-fold respectively, the Ras pathway may not be relevant. Taken together, this data suggests the p38/MAPK pathway may be a major driver of ER+ letrozole resistance. It was also noted that there was a 1.43-fold increase in p53 expression. This finding was not unusual as the T47D cell line expresses mutant p53 and in cultured cells, overexpression of tumor-associated p53 mutants has been shown to interfere with stress-induced apoptosis, increase resistance to chemotherapeutic drugs (8–12), promote genomic instability (13), and enhance proliferation (14).

Figure 3.

Figure 3.

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MAPK signaling cascade is induced in the letrozole-resistant T47DaromLR cells. Graph depict normalized mean pixel density of protein levels in lysates prepared from the T47Darom and T47DaromLR cells using the Human MAPK Phosphorylation Antibody Array Membrane where 17 different anti-MAPK related antibodies were spotted in duplicate, including 4 positive and 3 negative controls and 1 blank. Array signals were analyzed using Image Lab (BioRad) software. Values from duplicate spots were averaged and plotted. Results are expressed as the mean unit±SD (***p<0.001, **p<0.01, *p<0.05).

The p38/MAPK pathway drives letrozole resistance.

Since the proteomic analysis and the antibody array results demonstrated that increased p38/MAPK signaling was associated with the T47DaromLR cells, we wanted to determine whether inhibition of this pathway was responsible, in part, for the refractory nature of the cells as well as their aggressive phenotype. To test this, both the T47Darom and T47DaromLR cells were treated with 30 μM SB 203580 (a selective p38 MAPK) and proliferation was measured. Our results demonstrated that while the T47Darom cells were unaffected by SB 203580 treatment, there was a 1.47-fold (p<0.0001) reduction in the proliferation of the T47DaromLR cells (Figure 4). As the growth of the T47DaromLR cells was selectively decreased by the p38 inhibitor, we chose to assess whether this effect was associated with alterations in apoptosis. The caspase 3/7 assay was performed on both cell lines in the presence and absence of the p38 inhibitor (Figure 5). While the T47Darom cells were unresponsive, there was a slight induction in apoptosis in the T47DaromLR cells treated with the p38 inhibitor, suggesting the p38/MAPK pathway may play a role in endocrine resistance.

Figure 4.

Figure 4.

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Letrozole resistant cells are more sensitive to the growth inhibitory effects of p38/MAPK inhibitor SB 203508. The T47Darom cells were cultured in standard growth media and transferred to phenol red free media for two days prior to treatment with DMSO control or 30 μM of SB 203508 and cell proliferation assays were performed after 24 h. Proliferation was measured using the resazurin assay and graphs depict the fluorescence intensity of cells read at 430/560 nM wavelengths. Results are expressed as the mean unit±SD (****p<0.0001) of three independent experiments in triplicate; ns: not significant.

Figure 5.

Figure 5.

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Induction of apoptosis through targeting p38. The T47Darom cells were cultured in standard growth media and transferred to phenol red free media for two days prior to treatment with DMSO control or 10 μM of SB 203508 and caspase 3/7 activity was assessed by measuring the fluorescent intensity of cells at 530/502 nM wavelength. Results are expressed as the mean unit±SD (**p<0.01) of three independent experiments in triplicate.

Discussion

Aromatase inhibitors have been the first-line agents for postmenopausal women with hormone receptor positive metastatic breast cancer for many years. However, like many agents, some women develop resistance and stop responding to therapies, which can result in tumor relapse, metastasis, and a more aggressive phenotype. Thus, understanding mechanisms of resistance are critical to the development of novel strategies to address these patients. Previous reports by our group have identified a signature of letrozole resistance that was associated with estrogen independence, increased EGFR and HER2, enhanced motility, and an EMT phenotype (2). We focused on understanding the mechanisms of AI resistance associated with hormone refractory tumors, however the distinctions between AI-resistance in the hormone dependent versus hormone independent settings remain unclear.

Here, we utilized a non-migratory, ER+ letrozole resistant cell line (i.e., derived from the T47D epithelial cells) as a model system to initially characterize changes that occur as cells transition to an acquired AI-resistant phenotype. Although these cells are ER+, they are cross resistant to the selective estrogen receptor modulator tamoxifen, (3) further demonstrating their aggressive nature and limited therapeutic options. As the AI-sensitive cells transitioned to AI-resistance, both HER2 and MAPK expression were induced (6). This was a similar phenomenon previously observed in the hormone-independent AI-resistant LTLT-Ca cells (2). When the protein expression of a panel of phosphorylated receptor tyrosine kinases were evaluated, pErbB2 (i.e., pHER2) expression was decreased in the T47DaromLR cells compared to the T47DaromLR cells, suggesting that other pathways may be responsible for the acquired resistance. This was a unique feature of the T47DaromLR cells compared to the LTLT-Ca cells. Previous reports have demonstrated that increased pHER2 expression in the LTLT-Ca cells and trastuzumab was capable of reversing the resistance and restoring sensitivity to letrozole (15).

Since the T47DaromLR cells exhibited increased MAPK, we evaluated a panel of phosphorylated proteins involved in the MAPK signaling cascade. Signaling components and effectors of the p38/MAPK pathway (i.e., MKK6 and p38) were significantly increased while proteins involved in the PI3K/Akt/mTOR pathway (Akt and mTOR) were unchanged. It was interesting that signaling components of the Ras pathway (i.e., MEK and ERK1) were unchanged, while the downstream effectors (i.e., RSK1 and RSK2) of the Ras and p38/MAPK pathway were the predominant kinases that were increased. In general, RSK phosphorylates various substrates that control diverse cellular processes and when inhibited, it eliminates breast tumor initiating cells which are de novo chemo-resistant (16). Given these findings, it is likely that ERdependent AI resistance is mediated through the p38/MAPK signaling cascade. The observation that the MAPK signaling was increased in the T47DaromLR cells was not completely novel, since it has been previously reported that HER2, p-Shc, Grb2, and the signaling proteins in the MAPK cascade were increased in the LTLT-Ca cells (1).

A global proteomic analysis was conducted using the T47Darom and the T47DaromLR cells to further identify a protein signature specifically associated with ER+ AI resistance. Since we identified that acquired resistance was associated with the p38/MAPK signaling cascade, it is notable that one of the upstream signaling molecules, MAP3K6 was up-regulated. Out of a total of 127 significantly increased proteins, MAP3K6 exhibited the highest expression and was associated with decreased relapse-free survival. This further underscored the aggressive nature of breast tumors fitting this profile. Based on these findings, it is likely that as letrozole sensitive cells transition to a letrozole resistant phenotype they preferentially proliferate through activation of the p38/MAPK pathway inducing phosphorylation of RSK1/RSK2. Failure of these cells to respond to tamoxifen suggests they may utilize additional ligand independent signaling mechanism potentiated by p38. When the cells were treated with a p38 inhibitor, the T47Darom cells exhibited decreased proliferation and a slight induction in apoptosis.

While the major focus of this study was the p38/MAPK pathway, other proteins were identified including a protein disulfide isomerase (PDI) P4HB (prolyl 4-hydroxylase b). During times of oxidative stress or endoplasmic reticulum stress proteins unfold and molecular chaperones, like P4HB, assist in restoring unfolded proteins to their native conformation (17). A previous report has found that P4HB promoted a malignant phenotype in glioma cells via MAPK signaling (18) and identified P4HB as a novel target of chemoresistance in glioblastoma multiforme (19). It was hypothesized that perturbation of P4HB may sensitize the chemo-resistant glioma cells. While it is also known that PDIs serve as molecular chaperones to maintain ERα structure and function (20), the significance of high levels of P4HB and/or convergence with MAPK signaling pathways in AI-resistance remains unclear.

The proteomic analysis also revealed increased midasin (MDN1) expression which functions as a nuclear chaperone that is involved in the assembly/disassembly of macromolecular complexes in the nucleus, and is associated with maturation of 60S ribosome subunits (21). The finding that midasin was overexpressed in the T47DaromLR cells was interesting because a previous proteomic analysis demonstrated a 35-fold increase in MDN1 expression in LTLT-Ca cells cultured as mammospheres compared to LTLT-Ca cells cultured adherently (22, 23). While the role of midasin in breast cancer remains unclear, it is known that ribosomal biogenesis remains at the heart of translation. This process requires extensive regulation and coordination to meet the cellular demands of continuous ribosome production and aberrant midasin expression and subsequent interference with ribosomal biogenesis affects translation and the overall fitness of the cell.

While the proteomic signature of ER+ AI resistance is associated with enhanced p38/MAPK signaling there are additional factors contributing to this phenotype that are yet to be understood. Future studies will allow understanding how various signaling cascades converge to induce acquired letrozole resistance.

Acknowledgements

The Authors thank the ITT Research Institute and Dr. Akash Gupta for generously donating the T47Darom and T47DaromLR cell lines.

Funding

This work was supported in part by the National Institutes of Health [grant number 1SC1GM126617] awarded to Syreeta L. Tilghman. This publication was made possible by funding from the Louisiana Cancer Research Consortium and the National Institutes of Health Research Centers at Minority Institutes [grant numbers 8G12MD007595 and U54MD007582] from the National Institute on Minority Health and Health Disparities. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the Louisiana Cancer Research Consortium or the NIH.

Footnotes

Conflicts of Interest

The Authors declare no conflicts of interest in relation to this study.

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