FASN inhibition as a potential treatment for endocrine‑resistant breast
cancer
Aleksandra Gruslova1
· Bryan McClellan3
· Henriette U. Balinda1
· Suryavathi Viswanadhapalli2
· Victoria Alers1
Gangadhara R. Sareddy2
· Tim Huang1
· Michael Garcia1
· Linda deGrafenried3
· Ratna K. Vadlamudi2
Andrew J. Brenner1,4
Received: 27 January 2021 / Accepted: 14 April 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021
Abstract
Purpose The majority of breast cancers are estrogen receptor (ERα) positive making endocrine therapy a mainstay for
these patients. Unfortunately, resistance to endocrine therapy is a common occurrence. Fatty acid synthase (FASN) is a key
enzyme in lipid biosynthesis and its expression is commensurate with tumor grade and resistance to numerous therapies.
Methods The efect of the FASN inhibitor TVB-3166 on ERα expression and cell growth was characterized in tamoxifen￾resistant cell lines, xenografts, and patient explants. Subcellular localization of ERα was assessed using subcellular frac￾tionations. Palmitoylation and ubiquitination of ERα were assessed by immunoprecipitation. ERα and p-eIF2α protein levels
were analyzed by Western blotting after treatment with TVB-3166 with or without the addition of palmitate or BAPTA.
Results TVB-3166 treatment leads to a marked inhibition of proliferation in tamoxifen-resistant cells compared to the
parental cells. Additionally, TVB-3166 signifcantly inhibited tamoxifen-resistant breast tumor growth in mice and decreased
proliferation of primary tumor explants compared to untreated controls. FASN inhibition signifcantly reduced ERα levels
most prominently in endocrine-resistant cells and altered its subcellular localization. Furthermore, we showed that the
reduction of ERα expression upon TVB-3166 treatment is mediated through the induction of endoplasmic reticulum stress.
Conclusion Our preclinical data provide evidence that FASN inhibition by TVB-3166 presents a promising therapeutic
strategy for the treatment of endocrine-resistant breast cancer. Further clinical development of FASN inhibitors for endocrine￾resistant breast cancer should be considered.
Keywords Fatty acid synthase · Endocrine resistance · Breast cancer · FASN · Estrogen receptor · Endoplasmic reticulum ·
ER stress
Introduction
The past decade has seen signifcant advancement in increas￾ing survival in the management of estrogen receptor alpha
(ERα)-positive breast cancer. The use of selective estrogen
receptor downregulators and modulators (SERMs) (e.g.,
fulvestrant, tamoxifen) [1], mTOR inhibitors (e.g., everoli￾mus) [2], aromatase inhibitors (AI) [3], and most recently
cyclin-dependent kinase inhibitors (e.g., ribociclib, Palboci￾clib, and abemaciclib [4–6]) has helped to extend the overall
survival of the breast cancer patients. Yet, they do not ofer
an enduring cure and all patients will eventually succumb
to their disease. The American Cancer Society predicts that
over 42,000 women will die from breast cancer in 2020,
with the majority expressing the estrogen receptor (ERα) [7].
Furthermore, the latest generation of targeted therapies has
Aleksandra Gruslova and Bryan McClellan have contributed
equally to this work.
* Andrew J. Brenner
[email protected]
1 UT Health San Antonio MD Anderson Cancer Center,
San Antonio, TX, USA
2 Department of Obstetrics and Gynecology, University
of Texas Health San Antonio, San Antonio, TX, USA
3 The University of Texas At Austin, Austin, TX, USA
4 South Texas Research Facility, University of Texas
Health San Antonio, STRF 2.208.58403 Floyd Curl Dr,
San Antonio, TX 78229, USA
a higher level of toxicity relative to SERMs and AIs. Thus,
there is still a signifcant need to fnd additional treatment
options that can help to eliminate the mortality associated
with metastatic ERα-positive breast cancer, as well as to
identify new therapies that are more efective, less toxic,
and impact survival.
Alteration in endogenous lipid metabolism is an estab￾lished hallmark of cancer and contributes to an aggressive
and drug-resistant phenotype [1, 3, 8]. Among the enzymes
responsible for lipid biosynthesis are the ATP citrate lyase
(ACLY), acetyl-CoA carboxylase [8], stearoyl-CoA desatu￾rase (SCD), and fatty acid synthase (FASN) [1, 8]. Briefy,
endogenous synthesis of long-chain fatty acids, such as pal￾mitate, is an NADPH-dependent reaction that is mediated
through FASN using acetyl-CoA and malonyl-CoA as sub￾strates [9, 10]. Intracellular palmitate serves as a substrate
for both post-translation protein lipidation and membrane
phospholipid biosynthesis, while also participating as an
intracellular signaling molecule [8, 9]. FASN was initially
discovered as the oncogenic antigen-519 (OA-519) that was
highly expressed in aggressive breast cancer [11]. Moreover,
it was found that FASN inhibition using cerulenin leads to
an induction of apoptosis [11]. Since its discovery as an
oncogenic antigen, numerous preclinical and clinical thera￾pies have been developed to target its catalytic β-ketoacyl
reductase and thioesterase domains [9, 12, 13]. However,
early developed FASN inhibitors were limited to preclini￾cal use due to unwarranted side efects. Newer and more
targeted therapies have been developed with little to no side
efects [1]. TVB-2640 is a potent and reversible inhibitor
of the FASN enzyme that has been validated in multiple
tumor cell lines, as well as in clinical studies [12, 14, 15].
TVB-2640 inhibits the ketoacyl reductase (KR) enzymatic
activity of the FASN enzyme and is uncompetitive towards
both NADPH and Acetoacetyl-CoA in inhibiting KR activ￾ity. The activity of TVB-2640 was seen across several cell
lines, including MDA-MB-453, MDA-MB-231, MCF7,
and BT-474. The pharmacokinetics [16] and metabolism of
TVB-2640 have been examined with a series of in vitro and
in vivo studies, including toxicokinetic studies. Overall, the
PK properties determined to date for TVB-2640 demonstrate
that it is orally absorbed in humans and has a mean half-life
of approximately 10–13 h [16].
The safety and preliminary efcacy of TVB-2640 in a
total of 78 patients with multiple primary tumors across 11
centers [12] was recently completed. Pharmacokinetic analy￾ses showed a favorable profle with daily dosing, no attrib￾utable grade 3 adverse events at the recommended phase 2
dose of 100 mg/m2
, and excellent pharmacodynamics with
rapid reduction in both palmitate production and a reciprocal
increase in precursor malonyl-carnitine in serum. Overall,
5 confrmed RECIST partial responses (cPR) and multiple
patients with prolonged stable disease [17] (≥16 weeks)
were observed [18]. To preliminarily explore the activity
of TVB-2640 in taxane-resistant metastatic breast cancer, a
dose expansion cohort at the recommended phase 2 dose was
performed. Fifteen patients were enrolled, and represented
a heavily pretreated population with an average of 7 prior
regimens and all considered taxane-resistant [18]. Interest￾ingly, 2 of 3 patients with confrmed partial responses had
tamoxifen refractory ERα+disease, as did all patients with
stabilization beyond 24 weeks. While high FASN expres￾sion was relatively sensitive for predicting response, it was
by no means specifc as a signifcant percentage of patients
with low FASN expression had signifcant stabilization of
disease and included our longest responders. These fnd￾ings suggested the possibility of selective activity of FASN
inhibition in endocrine-resistant ERα+breast cancer that
was not specifcally dependent on the high expression of the
enzyme [18]. Mechanistic studies show that FASN inhibition
with TVB-3166, a TVB-2640 analog with a slightly lower
molecular weight for in vitro use, in a tumor cell-specifc
fashion disrupts lipid raft architecture, inhibits biological
pathways such as lipid biosynthesis, PI3K–AKT–mTOR
and β-catenin signal transduction, and inhibits expression
of oncogenic efectors such as c-Myc [12]. Previous studies,
including Menendez et al., have illustrated crosstalk between
FASN and ERα and that inhibition of FASN may lead to
the reduction of ERα protein, induction of CDK inhibitors
p21CIP1 and p27kip1, and suppression of estradiol-stimulated
proliferation and anchorage-independent colony formation
[1], 19, 20. Intriguingly, Zadra et al., observed a degrada￾tion of the androgen receptor (AR) in castration-resistant
prostate cancer upon treatment with the FASN inhibitor,
IPI-9119, that was mediated through endoplasmic reticulum
stress (EnRS) [21, 22]. Both reduction in total AR protein
and EnRS was ameliorated upon palmitate treatment [21],
highlighting the role of FASN inhibition in induced EnRS.
Though both preclinical and clinical studies have elucidated
mechanisms of FASN inhibition for the treatment of multi￾ple therapy-resistant tumors, there is a lack of investigation
on the potential for FASN inhibition as a therapy option for
endocrine therapy-resistant breast cancers.
Based upon our previous preliminary clinical data show￾ing that FASN inhibition is benefcial in metastatic breast
cancer patients, in this study, we demonstrate that inhibi￾tion of fatty acid synthase can improve outcomes in ERα-
positive, endocrine-resistant, metastatic breast cancer. Fur￾thermore, we show that FASN inhibition specifcally targets
ERα through the induction of EnRS in tamoxifen-resistant
breast cancer.
Materials and methods
Cell lines
Human breast cancer cells MCF7 and ZR75 were purchased
from the American Type Culture Collection (ATCC, Manas￾sas, VA). Tamoxifen-resistant MCF7/TamR and ZR75-
ESR537S mutant cells were described [23]. Each cell line
was maintained according to the routine protocol and tested
for mycoplasma using PCR Mycoplasma detection kit (Pro￾moCell, Germany). Additionally, the cell lines were authen￾ticated by STRF DNA profling at UTHSA and UT South￾western core facilities.
Circulating tumor cells
Circulating tumor cells were donated from a patient with a
failed response to aromatase inhibitor therapy with ERα-
positive breast cancer. Isolated tumor cells were grown in
short-term mammosphere culture prior to viability analysis.
About 8 mL of whole blood from patients was collected
in k2-EDTA (purple top), inverted 2×, and placed on ice
as described previously with some modifications [24].
Red blood cells were lysed by adding Cell Dilution Bufer
(v:v=1:6) (ScreenCell, Westford, MA) and incubated at
room temperature for 2 min. The majority of blood cells
are removed using microfltration (#CY4FC-V2, Screen￾Cell). The identifcation of circulating tumor cells is car￾ried out using immunofuorescence with anti-EpCAM-FITC
(clone VU-1D9, StemCell Technologies, Cambridge, MA)
and anti-CD45-PE (BD Bioscience). CD45-/EpCAM+cell
retained on microflters are isolated individually into culture
media.
In vitro proliferation assay
For in vitro drug screening, breast cancer cells were plated
into 24-well plates in growth medium (DMEM containing
10% FBS and 1% L-glutamine). After overnight incuba￾tion, cell plating medium was replaced with advanced MEM
containing 1% charcoal-stripped FBS, 1% L-glutamine,
and started treatment with TVB-3166 (200 nM), tamoxifen
(1 µM) or combination for 14 days. All experiments were
carried out using three biological replicates. Plates were
incubated, imaged, and analyzed by an automated Live￾Cell analysis system (IncuCyte). TVB-3166 was provided
by Sagimet Biosciences (formerly 3 V Biosciences).
In vivo therapeutics studies in xenograft models
All animal experiments were performed after obtain￾ing UTHSA IACUC approval and using methods in the
approved protocol. For xenograft tumor assays, tamoxifen￾resistant MCF7/TamR cells (5 × 106
) in 1:1 volume of
Matrigel Matrix were injected into the mammary fat pads
of 6-week-old SCID mice (n =6 per group). A 0.18-mg
sustained-release 17β-estradiol pellet was placed into the
contralateral fank before injection. When tumors reach
measurable size (~100–300 mm3), animals were randomized
into three treatment groups: TVB-3166 (60 mg/kg via oral
gavage daily), tamoxifen (4 mg/kg via subcutaneous injec￾tion daily), or a combination. The body weights and tumor
size were recorded three times a week for adverse toxic
efects. Tumor volume was determined by measurements
in two dimensions using calipers, with volumes defned as:
L× W2/2, where L is the longest dimension of the tumor
and W the shortest. At the end of the experiment, the mice
were euthanized, and the tumors were removed, weighed,
and processed for IHC staining.
Patient‑derived explants (PDEx) studies
UT Health Hospital (San Antonio, TX) provided written
consent allowing the use of discarded surgical samples
for research purposes according to an institutional board￾approved protocol (#20070684H). The protocol for an
ex vivo culture model of breast tumors was adopted from
published studies [23]. Briefy, de-identifed fresh tumor
samples were dissected into 1 mm3
specimens and placed
on hydrated gelatin sponges in explant media containing
5% CCS FBS at 37 °C. After 24 h, media were replaced
with new media (1% CCS FBS) containing vehicle or TVB
(200 nM) for 72 h. Representative tissues were fxed in 10%
formalin at 4 °C overnight and subsequently processed into
parafn blocks. Sections were stained with hematoxylin and
eosin and examined to confrm and quantify the presence/
proportion of tumor cells. Immunohistochemistry was then
performed.
Immunohistochemistry (IHC)
For xenograft and PDEx samples, 5 µm sections were used
for standard IHC protocol staining with ERα (1:50, Roche),
Ki67 (1:100, Vector Laboratories Burlingame, CA) antibody
in conjunction with proper protocol. A proliferative index
was calculated as the percentage of Ki-67-positive cells in
fve randomly selected microscopic felds at 40×per slide.
For subcellular localization of ERα, cultured cells were
fxed in 100% cold methanol and stained with ERα anti￾body (Abcam, #ab16660) at 1:50 dilution. Goat-anti-rabbit
IgG Alexa Fluor 488 (Abcam, #ab150077) was used as the
secondary antibody at 1:100 dilution. DAPI was used as a
nuclear counterstain. PBS instead of the primary antibody
was used as the secondary antibody only control.
Western blot analysis and subcellular fractionation
Cells were plated in T75 fask overnight in advanced MEM
medium with 5% CS FBS and 1% glutamine. The follow￾ing day medium was replaced for 1% CS FBS and TVB
(200 nM) was added. The cells were treated for 24 or 72 h.
Cells were lysed in RIPA bufer containing protein inhib￾itors. A total of 20 μg protein was resolved in 12% SDS
polyacrylamide gel, transferred onto a PVDF or nitrocel￾lulose membrane and blocked by 5% non-fat milk or BSA
(Bovine Serum Albumin) in TBS-T bufer for 1 h at room
temperature. Membrane was probed overnight at 4 °C with
primary antibody followed by 1 h incubation with secondary
antibody at room temperature. Signal detection was done
using ECL chemiluminescent kit (Invitrogen, #WP20005) or
West Femto Maximum Sensitivity Substrate (Thermo Sci￾entifc, #34095) with the membranes visualized on a Kodak
Image Station or Syngene Imager with GeneSnap software.
Primary antibodies used were GAPDH (1:1000), ERα_SP1
(1:500), p-eIF2α (1:1000, CST, #9727), eIF2α (1:1000, CST
#9722), ERα (1:1000, CST #8644 T), and secondary anti￾bodies used were anti-mouse-HRP (1:2000) and anti-rab￾bit-HRP (1:3000). Subcellular fractionation was performed
using a Subcellular Protein Fractionation kit (Thermo Scien￾tifc), according to the manufacturer’s instructions.
Palmitate‑BSA conjugation and cell treatments
Sodium palmitate (Millipore Sigma, #P9767) was heated
to 70 ̊C for 30 min in autoclaved H2O. Aqueous solution
of sodium palmitate was added dropwise to free fatty acid
(FFA)- stripped BSA solution and heated at 37 ̊C for 2 h
to fnalize conjugation to a 6:1 molar ratio of palmitate to
BSA. Prior to cell treatment, stock solutions of either FFA
stripped BSA (vehicle control) or Palmitate: BSA (6:1 molar
ratio) were heated to 37 C and added to 2% charcoal-stripped ̊
FBS media. MCF7 and MCF7/TamR cells were seeded in
six-well plates at a density of 350,000/well and allowed to
adhere for 24 h. 10% FBS DMEM media were replaced with
2% charcoal-stripped FBS media containing either BSA,
Palmitate:BSA, TVB-3166, TVB-3166+BSA, or TVB-
3166+Palmitate:BSA. Additionally, cells were pretreated
with 1,2-Bis(2-Aminophenoxy)ethane-N,N,N′,N′-tetraacetic
acid (BAPTA) (Millipore Sigma Aldrich, #A4926) for
60 min (20 μM) prior to the addition of TVB-3166 (200 nM)
for 72 h.
ERα palmitoylation
Detection of ERα palmitoylation was performed using
Acyl-Biotin Exchange (ABE) assay as previously
described [25]. Briefy, breast tumor cells were cultured
with or without TVB-3166 (200 nM) then lysed in ice-cold
lysis bufer containing 50 mM N-ethylmaleimide. BSA
assay was used to quantify the protein and 500 mg of pro￾tein was subjected to immunoprecipitation with protein
G sepharose beads (Millipore Sigma) using 1–5 mg of
antibody against ERα (Abcam). Each sample of beads
was split into two and incubated with or without hydrox￾ylamine (HAM). Selective labeling of palmitoylated
cysteine was performed using the bufer containing 3 mM
Biotin-BMCC (ProteoChem). The samples were boiled
at 80 °C for 10 min then resolved on SDS-PAGE gels.
Streptavidin-HRP (Invitrogen) at 1:5000 dilution was used
for detection of palmitoylated proteins.
RNA sequencing
RNA-seq was performed using the UTHSA core–estab￾lished protocol. Briefy, patient-derived explants were
treated with either vehicle or TVB (200 nM) for 72 h, and
total RNA was isolated using RNeasy mini kit (Qiagen)
according to the manufacturer’s protocol instructions.
NGS libraries were prepared from total RNA using Illu￾mina TruSeq stranded RNA Sample Prep kit following
manufacturer’s instructions and sequenced with 50 bp
single-read module using Illumina HiSeq 3000 system.
After adapter trimming and demultiplexing, short-read
sequence reads were processed using TopHat2 and HTSeq
for genome alignment and expression quantifcation. Dif￾ferential expression analysis was performed by DEseq
and signifcant genes with at least 1.5-fold change with
p < 0.01 were chosen for analysis. The interpretation of
biological pathways using RNA-seq data was performed
using gene set enrichment analysis. RNA-seq data have
been deposited in the GEO database under accession num￾ber: GSE166018.
Human phospho‑receptor tyrosine kinase array
MCF7/TamR cells were treated with either tamoxifen
(Tam, 1 mM), TVB-3166 (200 nM), or a combination for
72 h, then relative levels of RTK-phosphorylation were
determined using the Proteome Profler Human Phospho￾RTK Array kit following the manufacturer’s protocol
(R&D Systems). The phospho-RTK array membranes were
imaged using a Licor Fc imager with chemiluminescence.
Analysis was performed using Licor’s Studio Imager
Lite, where background-corrected mean pixel density is
depicted as signal.
Statistics
Data represented in the bar graphs are shown as
mean ± SE. T-test was performed for all comparisons.
Phospho-RTK array data were analyzed using Prism 8
software. A value of p < 0.05 was considered as statisti￾cally signifcant. RNA-seq data were analyzed using IPA
software.
Results
Inhibition of FASN blocks cell growth and cell cycle
progression in tamoxifen‑resistant breast cancer
cells through reduced ERα expression
The efects of FASN inhibition on the growth of tumor
cells have been confrmed in several breast cancer cell
lines including ER+cell lines MCF7 and ZR75, and tri￾ple-negative MDA-MB-231 cells. To ascertain whether
FASN inhibition can inhibit growth in endocrine-resistant
cells, we evaluated the proliferation rate of both paren￾tal MCF7 and their derivative from long-term exposure
to tamoxifen (MCF7/TamR). Cells were cultured with
either tamoxifen (Tam, 1 μM), TVB-3166 (200 nM), or
the combination. TVB-3166 is the preclinical version of
TVB-2640 with a slightly lower molecular weight and
increased solubility compared to TVB-2640 [12]. TVB-
3166 led to a signifcant growth inhibition in both MCF7
and MCF/TamR cell lines (Fig. 1a). Notably, TVB-3166
inhibited cell growth by 50% in MCF7/TamR cells as
compared to 20% in the parental MCF7 cells. Moreo￾ver, when TVB-3166 was combined with Tam, the com￾bination resulted in complete growth inhibition of both
Tam-sensitive and Tam-resistant MCF7 cells. This was
associated with fow cytometry cell cycle analysis with
propidium iodide DNA staining. A substantial increase of
S phase population at 72 h is suggestive of DNA-damage
or replicative stress (Fig. 1b). Western blot analysis of
ERα protein levels showed a time-dependent loss of pro￾tein expression after TVB-3166 treatment in MCF7 and
MCF7/TamR cells, which was markedly greater in MCF7/
Fig. 1 Therapy-resistant breast cancer cells have greater sensitiv￾ity to FASN inhibition than parental breast cancer cells. a Kinetics
of cell growth of either MCF7 (upper) or tamoxifen-resistant MCF7
(lower) cells when exposed to vehicle control (blue), 1  μM tamox￾ifen (Tam) (red), 200  nM of TVB-3166 (green), or both (purple).
****p<0.0001. b Comparison of log-scale histograms of propidium
iodide-stained cells. Controls were untreated cells. c Western blot
analysis of ERα from parental and Tamoxifen-resistant MCF7 cells
after 30 and 72 h of exposure to 200 nM of TVB-3166 compared to
the control. d Western blot analysis of ERα from estrogen receptor 1
(ESR1)-mutant ZR75 cells after 30 and 72 h of exposure to 200 nM
of TVB-3166 compared to control. E) Viability of aromatase inhibi￾tor-resistant circulating tumor cells (CTCs) cultured and treated with
200 nM of TVB-3166
TamR cells (Fig. 1c). Following 72 h of exposure, protein
levels decreased by 90% in the MCF7/TamR cells, while
only 20% in the parental cells. To evaluate the association
of FASN inhibition with ERα, we tested the efect of TVB-
3166 on breast cancer cells with mutant estrogen receptor
alpha gene (ESR1). In particular, TVB-3166 signifcantly
inhibited ERα expression in ZR75-mutY537S cells com￾pared to the control (Fig. 1d). ERα protein decreased by
70% and 90% after 24 and 72 h, respectively. Using cir￾culating tumor cells (CTCs), we evaluated the efect of
FASN inhibition on primary tumor cultures. CTCs were
collected from a patient with from a patient with ESR1
D538G who had failed four lines of endocrine therapy
(including tamoxifen, letrozole, fulvestrant, and combina￾tions with PI3K and CDK inhibitors) and established into a
short-term mammosphere culture. Fig. 1e shows that after
72 h of TVB exposure (200 nM), there was a 50% decrease
in cell viability (p<0.05), confrming our in vitro results.
These data support that FASN inhibition with TVB-3166
is associated with ERα signaling pathways.
TVB‑3166 inhibits growth of tamoxifen‑resistant
breast tumors in mice
In vivo efcacy of FASN inhibition alone and in combi￾nation with tamoxifen on breast tumor growth was investi￾gated using the MCF7/TamR xenograft model that exhibits
resistance to tamoxifen [26–28]. Upon noticeable tumor
size (100–300 mm2
), nude mice bearing MCF7/TamR cells
were randomized in 3 treatment groups: tamoxifen (100 μg/
mice, subcutaneous, control), TVB-3166 (60 mg/kg, oral
gavage) or combination. Eight weeks of treatment with TVB
alone or in combination with tamoxifen resulted in statis￾tically signifcant inhibition of MCF7 tamoxifen-resistant
xenografts growth (n=6 mice/group) compared to tamox￾ifen alone (Fig. 2a). As shown in (Fig. 2b), the average size
of the tumors in the combination group was 50% and 30%
smaller compared with tamoxifen or TVB groups, respec￾tively. Animals tolerated the treatment very well without any
efect on their weight. These data indicate that TVB-3166 is
a potent inhibitor of the growth of endocrine-resistant breast
Fig. 2 TVB-3166 inhibits tumor growth of tamoxifen-resistant MCF7
xenografts. a Tumor growth curve of MCF7/TamR during treatment.
b Weights of extirpated tumor xenografts posttreatment. Tam vs
TVB+Tam, p value=0.047; TVB vs TVB+Tam, p value=0.038. c
Immunohistochemistry analysis of tumors shows signifcant decrease
in ERα after TVB-3166 treatment alone (p value=0.002) or in com￾bination with tamoxifen (p value=0.003). Images were taken with
40X magnifcation. Scale bar=0.2 cm. d Quantifcation of data in c
tumors in vivo with no overt signs of toxicity in mice. Fur￾thermore, immunohistochemistry analysis of tumors showed
signifcant decrease of ERα levels after TVB treatment alone
or in combination with tamoxifen (p=0.002 and p=0.003,
respectively) (Fig. 2c and d).
TVB‑3166 inhibits proliferation in patient‑derived
primary tumor explants
Given potential diferences between in vitro and in vivo, we
used an ex vivo culture model of primary breast tumors to
characterize changes associated with FASN inhibition. This
model maintains the native tissue architecture and critical
cell-to-cell signaling of the tumor microenvironment and
thus better recapitulates the complexity and heterogeneity
of human breast cancer in a laboratory setting. Addition￾ally, these ex vivo techniques allow quantifcation of clini￾cally relevant endpoints that are more applicable to patient
treatment than established cell lines. In brief, fresh tumor
tissue was obtained from surgical resection or biopsy, dis￾sected into 1 mm3
specimens, and placed on hydrated gelatin
sponges in explant media at 37 °C. After 24 h, new media
containing vehicle or investigational agents was added
(Fig. 3a). Four ER+, HER2− breast tumor specimens taken
from treatment naïve patients were cultured in the absence
or presence of TVB-3166 (200 nM) for 72 h. Immunohis￾tochemistry analysis indicated a decrease in proliferation
after TVB treatment. Ki67 staining was signifcantly lower
in treated tumors compared to untreated (14% vs 36%,
p<0.001) (Fig. 3b). Furthermore, FASN inhibition leads to
a signifcant reduction in the ERα levels in tumors (p<0.01)
(Fig. 3c).
Identifcation of diferentially expressed genes
in response to TVB‑3166 treatment
To investigate specifc biological outcomes from treatment in
tamoxifen-resistant breast cancer, RNA sequencing and phos￾pho-receptor tyrosine kinase (RTK) array were performed in
response to treatment with TVB-3166, tamoxifen, or in com￾bination. RNA sequencing analyses revealed that TVB signif￾cantly altered the mRNA expression of 219 genes (p<0.01)
in primary patient-derived breast tumor explants compared to
vehicle control (n=2) (Fig. 4a). Gene set enrichment analysis
(GSEA) of the diferentially expressed genes ranked by fold
change showed 2 enriched gene sets: (1) upregulation of genes
downregulated in ESR1-positive breast tumors compared to
the ESR1-negative tumors (Fig. 4b) and (2) downregulation of
genes involved in invasiveness signature resulting from can￾cer cell-microenvironment interaction (Fig. 4c). Additionally,
the phosphorylated-RTK assay in MCF7 and MCF7/TamR
cells revealed eight altered (p<0.01) phosphorylation levels
of RTKs afected in response to a combination of TVB-3166
and tamoxifen compared to tamoxifen alone. Of the eight, the
most dramatic changes were in the EGFR, FGFR2a, Tie2, and
ROR2 phosphorylation. TVB-3166 and tamoxifen combina￾tion treatment increased the phosphorylation of ROR2 and
EGFR dramatically compared to treatment with tamoxifen
alone (Fig. 4d).
TVB‑3166‑induced ERα degradation
in endocrine‑resistant breast cancer is mediated
through enhanced endoplasmic reticulum stress.
ERα is a nuclear ligand-activated transcription factor and
also an extrinsic plasma membrane receptor. ERα resides
Fig. 3 TVB-3166 inhibits proliferation and reduces ERα expression
in patient-derived primary tumor explants. a Schematic represen￾tation of ex  vivo culture model. b Immunohistochemistry analysis
shows that TVB-3166 treatment leads to a decrease in proliferation,
demonstrated by a reduction in Ki67 expression. Images were taken
with 20× magnifcation. Scale bar=50 μm. c Immunohistochemistry
shows a reduction in ERα expression upon treatment with TVB-3166
of the patient-derived primary tumor explants
Fig. 4 Identifcation of diferentially expressed genes between
vehicle-treated and TVB-3166-treated patient-derived primary
tumor explants, 564 and 518. a Heatmap representing diferentially
expressed genes between vehicle and TVB-3166. b and c Representa￾tive gene set enrichment analysis (GSEA) plots showing 2 enriched
gene sets: genes downregulated in ESR1-positive breast tumors
compared to ESR1-negative tumors (positive correlation) (b) and
invasiveness signature resulting from cancer cell-microenvironment
interaction (negative correlation) (c). d Representative bar graph of
phospho-receptor tyrosine kinase (RTK) assay showing statistically
signifcant changes in protein phosphorylation in MCF7/TamR cells
treated with 200 nM of TVB-3166, 1 μM Tamoxifen, or both. Rela￾tive pixel density is calculated relative to the corresponding untreated
(vehicle) controls. Phosphorylation of ROR2 and EGFR was the most
increased in response to combination treatment. ****p<0.0001;
***p<0.0005; **p<0.005; *p<0.05
Fig. 5 TVB-3166 alters subcellular localization of ERα and induces its
degradation in tamoxifen-resistant breast cancer. a The pie charts show
subcellular localization of ERα in MCF7 and MCF7/TamR cells deter￾mined by Western blotting. ERα expression in the cytoplasm is signif￾icantly more in MCF7/TamR cells compared to parental MCF7 cells
(p value=0.025). b Bar graphs represent degradation of ERα protein
in MCF7/TamR cells determined by Western blotting. TVB-3166 and
tamoxifen combination treatment leads to increased ERα degradation
predominantly in the nucleus, while unbound ERα shut￾tles between the cytoplasm and nucleus. To confrm that
long-term tamoxifen treatment causes translocation of ERα
out of the nucleus to the cytoplasm [29], we extracted ERα
from three diferent cell fractions (cytoplasm, membrane and
nucleus) of MCF7/TamR cells and its parental MCF7 cell
line. Western blot data showed that in MCF7 cells, more
than 50% ERα was in the nucleus, whereas in MCF7/TamR
cells, ERα level in cytoplasm was signifcantly higher com￾pared to the parental cell line (p=0.025) (Fig. 5a and Fig.
S1a). Moreover, there was an appreciable degradation of
total ERα protein in MCF7/TamR cells in response to TVB
and tamoxifen combination treatment (Fig. 5b and Fig. S1b).
In order to understand the localization and levels of ERα in
response to TVB-3166 treatment, we investigated the levels
of palmitoylation and ubiquitination of the ERα in MCF7/
TamR cells. However, we observed no major diferences
in TVB-3166- treated and untreated samples in the levels
of palmitoylated and ubiquitinated ERα protein (Fig. S2).
We also investigated the involvement of lysosomes in TVB-
3166-dependent ERα degradation by pretreating the cells
with chloroquine, a drug that inhibits lysosomal enzymes by
changing endosomes and lysosomes internal pH [30]. Our
data showed that there was increased lysosomal degrada￾tion in the presence of TVB-3166, which was unexpected
(Fig. S3).
Alterations in membrane lipid composition are known to
result in the induction of EnRS [31] (Fig. 6a). In periods of
EnRS, the protein kinase R (PKR)-like endoplasmic reticu￾lum kinase (PERK) is activated resulting in serine phospho￾rylation of the eukaryotic initiation factor-2 complex in the
alpha subunit (eIF2α) [32]. This phosphorylation results in
a competitive inhibition of the guanine nucleotide exchange
factor for its beta-subunit (eIF2β), ultimately leading to an
inhibition of protein translation [33]. Additionally, previous
studies have elucidated the role of FASN inhibition lead￾ing to EnRS in prostate cancer that was attenuated upon
treatment with palmitate, the major product of FASN [21].
To investigate the efects of FASN inhibition and EnRS
induction in tamoxifen-resistant breast cancer, both MCF7/
TamR and MCF7 cells were subjected to treatment with or
without 200 nM of TVB in combination with either pal￾mitate or BSA. Palmitate was conjugated to BSA in a 6:1
molar ratio, to be transported into the cell. Treatment with
TVB-3166 resulted in a decrease in total ERα protein that
was only apparent in MCF7/TamR cells. Additionally, the
Fig. 6 TVB-3166 induces endoplasmic reticulum stress (EnRS) that
targets ERα in tamoxifen-resistant breast cancer. a Proposed model
of TVB-3166 induced ERα degradation in tamoxifen-resistant breast
cancer. b Western blot analysis of MCF7 and MCF7/TamR cells
treated with 200  nM TVB-3166 for 72  h, or TVB-3166 with either
palmitate (P) or BSA (B). Palmitate was conjugated to BSA in a
6:1 molar ratio. Bands were quantifed using area under the curve
analysis with NIH ImageJ software. Graphs represent an average
of 3 independent experiments. ERα was standardized to GAPDH,
while p-eIF2α was standardized to total eIF2α. **Indicates a statis￾tical change in ERα/GAPDH between TVB-3166 and TVB+PAL￾MITATE-treated group (p=0.02) *Indicates statistical change of
p-eIF2α/eIF2α between TVB-3166 and TVB-3166+PALMITATE
group (p=0.05). c Western blot analysis of MCF7 and MCF7/TamR
cells. Both cell lines were pretreated with EnRS inhibitor, BAPTA,
for 60 min prior to treatment with TVB-3166 in order to compare to
the efects of TVB+PALMITATE
decrease in ERα expression was accompanied by an increase
in EnRS, as indicated by the levels of phosphorylated-eIF2α
(p-eIF2α) (Fig. 6b). Moreover, the addition of palmitate res￾cued the expression of ERα, while ameliorating markers of
EnRS (p-eIF2α) (Fig. 6b). The quantifcation of band inten￾sity revealed a statistically signifcant (p=0.02) increase in
ERα protein and decrease in p-eIF2α (p≤0.05), upon the
addition of palmitate when treated with TVB-3166 com￾pared to TVB-3166 alone (Fig. 6b). Considering the addition
of palmitate attenuated stress in response to FASN inhibi￾tion, we sought to determine if the use of an EnRS inhibitor
would have similar efects upon treatment with TVB-3166.
Intriguingly, pretreating with the EnRS inhibitor, BAPTA,
prior to the addition of TVB-3166 for 72 h, rescued ERα
expression while lowering EnSR (p-eIF2α) similar to that
of the TVB+palmitate treatment (Fig. 6c). This rescuing
efect of ERα along with the decrease in EnRS was only
demonstrated in tamoxifen-resistant cells and not tamoxifen
sensitive, indicating a cell type-specifc response.
Discussion
Estrogen-positive breast cancer accounts for nearly 70% of
breast cancer-related deaths and often is treated with estro￾gen receptor antagonists such as tamoxifen. Unfortunately,
ERα targeted therapies can result in resistance, subsequent
metastasis, and death. Reprogramming of lipid metabo￾lism is an established hallmark of cancer and is associated
with drug resistance, including resistance to anti-endocrine
therapies. The rate-limiting enzyme for endogenous lipid
biosynthesis, fatty acid synthase (FASN), is overexpressed
in numerous cancers and is associated with drug resistance.
The FASN inhibitor TVB-2640 has shown preliminary evi￾dence of activity in a phase 1 clinical trial dose expansion
arm for metastatic breast cancer. In this study, we illus￾trate that FASN inhibitor, TVB-3166 (preclinical version
of TVB-2640), signifcantly reduces growth and prolifera￾tion of tamoxifen-resistant breast cancer in in vitro, ex vivo,
and in vivo models by inducing ERα degradation through
increased endoplasmic reticulum stress.
The inhibition of FASN has several consequences that
result in either apoptosis or stasis in cell progression [12].
The primary product of FASN includes the long-chain fatty
acid, palmitate, that can post-translationally modify onco￾proteins, such as Wnt and epidermal growth factor receptor
(EGFR) that facilitate their intracellular localization and
membrane hydrophobicity [34]. Moreover, palmitate itself
undergoes subsequent alterations to generate phospholipids
for membrane synthesis, which is essential for rapidly prolif￾erating tissue [35]. A phase 1 study demonstrated a decrease
in circulating palmitate metabolites in patients treated with
TVB-2640 [18]. FASN is transcriptionally and post-tran￾scriptionally regulated through PI3K/Akt/mTOR pathway
downstream of receptor tyrosine kinases (RTKs), such as
EGFR [36]. Thus, FASN-induced palmitoylation of RTKs
could mediate a feedforward loop to support lipid metabolic
programming in anti-endocrine resistance. Additionally, we
have also observed an increase in malonyl-carnitine, a more
stable derivative of the FASN substrate malonyl-CoA [18].
Malonyl-CoA has a short half-life and is therefore difcult
to measure. Thus, the decreased cellular pool of palmitate
for membrane synthesis and post-translational modifcations
could explain the growth inhibitory efects of FASN inhibi￾tion observed.
In response to treatment with tamoxifen, TVB-3166, or
in combination, RNA sequencing and phospho-receptor
tyrosine kinase arrays demonstrated a signifcant increase in
the receptor tyrosine kinase-like orphan receptor-2 (ROR2)
when treated with the combination of TVB and tamoxifen
compared to tamoxifen treatment alone in tamoxifen-resist￾ant cells. Classically, both ROR1 and ROR2 are involved
in neurogenesis and embryonic development. Moreover,
expression of ROR1 and ROR2 is associated with increased
invasion and poor prognosis in numerous cancers [37, 38].
Interestingly, in certain cancers, ROR2 expression exhib￾its tumor-suppressing characteristics. Recent studies have
illustrated that ROR2 hypermethylation in nasopharyngeal,
esophageal, and breast cancer cell lines promoted EMT
and proliferation [37]. Additionally, ROR2 overexpression
resulted in reduced Akt and β-catenin signaling in multiple
cancer cell lines [37]. Not only does ROR2 possess tumor￾suppressing traits but is also implicated in inducing apopto￾sis through the EnRS pathway. Recently, ROR2 promoted
apoptosis in ovarian carcinoma through the induction EnRS
sensing protein, inositol requiring enzyme-1α (IRE1α), and
downstream C/EBP homologous protein (CHOP) [37, 38].
Thus, ROR2 overexpression observed in response to TVB
and tamoxifen treatment could be illustrating tumor-sup￾pressing characteristics.
Additionally, we demonstrate that treatment with TVB-
3166 leads to a decrease in ERα protein that was specifc to
tamoxifen-resistant breast cancer compared to the tamox￾ifen sensitive. Several studies have elucidated an increase
in cap-dependent mRNA translation through mTOR and
ERK pathways as a mechanism of acquired tamoxifen resist￾ance. Moreover, FASN inhibition has been demonstrated to
attenuate Akt and mTORC1 signaling in multiple studies. In
our lab, we observed an inhibition in the phosphorylation of
Akt, which is upstream of mTORC1. Pathways upregulated
in tamoxifen resistance, such as mTOR, involve the phos￾phorylation of translational inhibitory complexes such as
4E binding proteins (4E-BP1) leading to an alleviation of
eIF4E cap-binding protein. The mRNA translational initia￾tion complex is composed of several eIF4F family members
that include an RNA helicase (eIF4A), scafolding protein
(eIF4G), and the rate-limiting cap-binding protein (eIF4E)
[32, 33]. Moreover, many oncoproteins and growth factors
such as cyclin D1 and vascular endothelial growth factor
(VEGF) are encoded by mRNAs with longer than average 5′
untranslated regions (UTRs) [32]. Translational complexes,
such as eIF4E, have an enhanced afnity for longer UTRs.
More specifcally, using a mutated eIF4E that renders it una￾ble to become phosphorylated resulted in an attenuation in
proliferation in response to tamoxifen in tamoxifen-resistant
cell lines [33]. This upregulation in mRNA cap-dependent
translation pathways in tamoxifen resistance holds the prem￾ise for an increased sensitivity to FASN inhibition in tamox￾ifen-resistant compared to tamoxifen-sensitive breast cancer.
We observed that FASN inhibition-induced ERα degra￾dation is mediated through the EnRS pathway. Historically,
higher ratios of phosphatidylcholine to phosphoethanola￾mine as well as increases in intracellular cholesterol lead to
EnRS [22]. Studies have shown alterations in intracellular
membrane lipids as well as a compensatory increase in cho￾lesterol biosynthesis in response to FASN inhibition. Recent
studies, conducted by Zadra et al., 2019, illustrated that
FASN inhibition in prostate cancer leads to an alteration in
phospholipids within the EnR membrane [21]. The initiation
of protein translation involves both the recognition of the
start codon in mRNA by the preinitiation complex as well as
the recruitment of various eukaryotic initiation factors (eIFs)
[33]. eIFs 1, 1A, 2, 3, 4, and 5 are all involved in translation
[33]. Moreover, the eIF2 complex is the site of translational
control in periods of starvation, stress, or viral infection [33,
39]. EnRS through the PKR like endoplasmic reticulum
kinase (PERK) results in the phosphorylation of eukaryotic
initiation factor 2α [22]. This results in protein translation
inhibition, which could explain the decrease in ERα protein
levels observed upon FASN inhibition. This EnRS response
to FASN inhibition was only observed in tamoxifen-resistant
breast cancer; however, the specifc response could be due to
the increased reliance upon translational machinery that has
been recorded previously in acquired tamoxifen resistance
[32, 33]. Intriguingly, the increased expression of ROR2
recorded in the phospho-RTK assay could be a potential
mediator of endoplasmic reticulum stress. Previous studies
connecting ROR2 expression to CHOP-induced apoptosis
highlight a potential connection to the PERK/p-eIF2α EnRS
pathway [38]. Not only does p-eIF2α lead to a decrease in
protein translation, but also allows the initiation complex
to continue down the mRNA until it reaches the alternate
reading frame [33]. The alternate reading frame of mRNA
is the location of translation for EnRS response proteins,
such as CHOP and ATF-4 [32, 33]. Thus, the overexpression
of ROR2 from treatment with TVB and tamoxifen could
potentially be contributing to endoplasmic reticulum stress.
We previously preliminarily demonstrated in a phase 1
study that FASN inhibition is a viable treatment option for
ER+breast cancer patients that have become resistant to
anti-endocrine therapies [18]. In this study, we explore the
potential mechanism and have shown that FASN inhibitor,
TVB-3166, targets ERα protein for degradation in tamox￾ifen-resistant breast cancer. This loss in ERα protein was
found to be as a result of increased endoplasmic reticulum
stress. Future studies should investigate the connection to
lipid metabolic programming and the mRNA translational
network. Moreover, more specific mechanisms linking
FASN expression directly to acquired anti-estrogen resist￾ance should be conducted. Finally, more investigation should
be done illustrating the role of ROR2 in tamoxifen-resistant
breast cancer. With more targeted and less toxic therapy
options, patients are extending not only their life, but their
quality of life. FASN inhibition, either as a monotherapy
or in combination, could be a potential novel treatment for
anti-endocrine therapy-resistant breast cancer.
Supplementary Information The online version contains supplemen￾tary material available.
References
1. Menendez J, Lupu R (2017) Fatty acid synthase regulates estro￾gen receptor-α signaling in breast cancer cells. Oncogenesis
6(2):e299–e299
2. Robertson JF et al (2016) Fulvestrant 500 mg versus anastrozole 1
mg for hormone receptor-positive advanced breast cancer (FAL￾CON): an international, randomised, double-blind, phase 3 trial.
The Lancet 388(10063):2997–3005
3. Lupu R, Menendez JA (2006) Targeting fatty acid synthase in
breast and endometrial cancer: an alternative to selective estrogen
receptor modulators? Endocrinology 147(9):4056–4066
4. Dickler MN et al (2017) MONARCH 1, a phase II study of
abemaciclib, a CDK4 and CDK6 inhibitor, as a single agent, in
patients with refractory HR+/HER2− metastatic breast cancer.
Clin Cancer Res 23(17):5218–5224
5. Hortobagyi GN et  al (2016) Ribociclib as first-line ther￾apy for HR-positive, advanced breast cancer. N Engl J Med
375(18):1738–1748
6. De Angelis C, Ignatiadis M, Piccart-Gebhart M (2020) Cyclin￾dependent kinases 4 and 6 inhibitors (CDK4/6i) versus chemo￾therapy in luminal B early breast cancer: lessons from the COR￾ALLEEN trial. Annals of Translational Medicine 8(21):1338
7. Desantis CE et al (2019) Breast cancer statistics. CA Cancer J Clin
69(6):438–451
8. Jones SF, Infante JR (2015) Molecular pathways: fatty acid syn￾thase. Clin Cancer Res 21(24):5434–5438
9. Menendez JA, Lupu R (2007) Fatty acid synthase and the
lipogenic phenotype in cancer pathogenesis. Nat Rev Cancer
7(10):763–777
10. Corominas-Faja B et al (2017) Clinical and therapeutic relevance
of the metabolic oncogene fatty acid synthase in HER2+ breast
cancer. Histol Histopathol 32(7):687
11. Kuhajda FP et al (1994) Fatty acid synthesis: a potential selec￾tive target for antineoplastic therapy. Proc Natl Acad Sci
91(14):6379–6383
12. Ventura R et al (2015) Inhibition of de novo palmitate synthesis by
fatty acid synthase induces apoptosis in tumor cells by remodeling
cell membranes, inhibiting signaling pathways, and reprogram￾ming gene expression. EBioMedicine 2(8):808–824
13. Patel M et al (2015) Abstract CT203: Report of a frst-in-human
study of the frst-in-class fatty acid synthase (FASN) inhibitor
TVB-2640. Can Res 75(15):CT203
14. Heuer TS et al (2016) FASN inhibition and taxane treatment
combine to enhance anti-tumor efcacy in diverse xenograft
tumor models through disruption of tubulin palmitoylation and
microtubule organization and FASN inhibition-mediated efects
on oncogenic signaling and gene expression.
15. Brenner A et al (2015) First-in-human study of the frst-in-class
fatty acid synthase (FASN) inhibitor, TVB-2640 as monotherapy
or in combination-fnal results of dose escalation.
16. Falchook G et al (2017) Abstract CT153: frst in human study of
the frst-in-class fatty acid synthase (FASN) inhibitor TVB-2640.
AACR.
17. WäsTER P et al (2011) Ultraviolet exposure of melanoma cells
induces fbroblast activation protein-α in fbroblasts: implications
for melanoma invasion. Int J Oncol 39(1):193–202
18. Brenner A et al (2017) Abstract P6-11-09: heavily pre-treated
breast cancer patients show promising responses in the frst in
human study of the frst-In-class fatty acid synthase (FASN)
inhibitor, TVB-2640 in combination with paclitaxel. AACR.
19. Cariou S et al (2000) Down-regulation of p21WAF1/CIP1 or
p27Kip1 abrogates antiestrogen-mediated cell cycle arrest in
human breast cancer cells. Proc Natl Acad Sci 97(16):9042–9046
20. Doisneau-Sixou S et al (2003) Estrogen and antiestrogen regula￾tion of cell cycle progression in breast cancer cells. Endocr Relat
Cancer 10(2):179–186
21. Zadra G et al (2019) Inhibition of de novo lipogenesis targets
androgen receptor signaling in castration-resistant prostate cancer.
Proc Natl Acad Sci 116(2):631–640
22. Little JL et al (2007) Inhibition of Fatty Acid Synthase Induces
Endoplasmic Reticulum Stress in Tumor Cells. Cancer Res
67(3):1262–1269
23. Viswanadhapalli S et al (2019) Estrogen receptor coregulator
binding modulator (ERX-11) enhances the activity of CDK4/6
inhibitors against estrogen receptor-positive breast cancers. Breast
Cancer Res 21(1):1–15
24. Chen CL et al (2013) Single-cell analysis of circulating tumor
cells identifes cumulative expression patterns of EMT-related
genes in metastatic prostate cancer. Prostate 73(8):813–826
25. Brigidi GS, Bamji SX (2013) Detection of protein palmitoylation
in cultured hippocampal neurons by immunoprecipitation and
acyl-biotin exchange (ABE). JoVE Journal of Visualized Experi￾ments 72:e50031
26. Nair BC et al (2011) Roscovitine confers tumor suppressive
efect on therapy-resistant breast tumor cells. Breast Cancer Res
13(3):R80
27. Wu Y et al (2018) Tamoxifen resistance in breast cancer is regu￾lated by the EZH2-ERalpha-GREB1 transcriptional axis. Cancer
Res 78(3):671–684
28. Viswanadhapalli S et al (2019) Estrogen receptor coregulator
binding modulator (ERX-11) enhances the activity of CDK4/6
inhibitors against estrogen receptor-positive breast cancers. Breast
Cancer Res 21(1):150
29. Fan P et al (2007) Long-term treatment with tamoxifen facili￾tates translocation of estrogen receptor α out of the nucleus and
enhances its interaction with EGFR in MCF-7 breast cancer cells.
Can Res 67(3):1352–1360
30. Steinman RM et al (1983) Endocytosis and the recycling of
plasma membrane. J Cell Biol 96(1):1–27
31. Fu S et al (2011) Aberrant lipid metabolism disrupts calcium
homeostasis causing liver endoplasmic reticulum stress in obesity.
Nature 473(7348):528–531
32. Gong C et al (2020) Phosphorylation independent eIF4E transla￾tional reprogramming of selective mRNAs determines tamoxifen
resistance in breast cancer. Oncogene 39(15):3206–3217
33. Geter PA et al (2017) Hyperactive mTOR and MNK1 phospho￾rylation of eIF4E confer tamoxifen resistance and estrogen inde￾pendence through selective mRNA translation reprogramming.
Genes Dev 31(22):2235–2249
34. Bollu LR et al (2015) Intracellular activation of EGFR by fatty
acid synthase dependent palmitoylation. Oncotarget 6(33):34992
35. Röhrig F, Schulze A (2016) The multifaceted roles of fatty acid
synthesis in cancer. Nat Rev Cancer 16(11):732
36. Lee G et al (2017) Post-transcriptional regulation of de novo lipo￾genesis by mTORC1-S6K1-SRPK2 Signaling. Cell 171(7):1545-
1558.e18
37. Li L et al (2014) Epigenetic identifcation of receptor tyrosine
kinase-like orphan receptor 2 as a functional tumor suppressor
inhibiting β-catenin and AKT signaling but frequently methylated
in common carcinomas. Cell Mol Life Sci 71(11):2179–2192
38. Li R et al (2019) ROR2 induces cell apoptosis via activating
IRE1α/JNK/CHOP pathway in high-grade serous ovarian carci￾noma in vitro and in vivo. J Transl Med 17(1):1–17
39. Fagan DH et al (2017) Acquired tamoxifen resistance in MCF-7
breast cancer cells requires hyperactivation of eIF4F-mediated
translation. Hormones and Cancer 8(4):219–229
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.