MOLECULAR AND CLINICAL ONCOLOGY 13: 70, 2020

HER2‑positive breast cancer that resists therapeutic drugs
and ionizing radiation releases sphingomyelin‑based
molecules to circulating blood serum
SATORU MONZEN1, YOTA TATARA2, YASUSHI MARIYA3, MITSURU CHIBA4,ANDRZEJ WOJCIK5 and LOVISA LUNDHOLM5
1

Department of Radiation Science, Hirosaki University Graduate School of Health Sciences, Hirosaki, Aomori 036‑8564;
2
Department of Glycotechnology, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori 036‑8562;
3
Department of Radiology, Mutsu General Hospital, Mutsu, Aomori 035‑0071; 4Department of
Bioscience and Laboratory Medicine, Hirosaki University Graduate School of Health Sciences, Hirosaki,
Aomori 036‑8564, Japan; 5Centre for Radiation Protection Research, Department of Molecular Biosciences,
The Wenner‑Gren Institute, Stockholm University, 114 18 Stockholm, Sweden
Received February 17, 2020; Accepted June 16, 2020
DOI: 10.3892/mco.2020.2140

Abstract. Breast cancer is the second most common cancer

in the world based on incidence, reaching more than 2 million
new cases in 2018, while continuing to increase. Invasive ductal
carcinoma is the most common type of this cancer, making
up approximately 70‑80% of all breast cancer diagnoses. In
particular, the type of breast cancer overexpressing humanepidermal growth factor receptor 2 (HER2) has potential ofstrong proliferation, migration and invasion and early treatment
is necessary. The authors identified and studied a single patient
displaying complete therapeutic resistance to monoclonalanti‑HER2 antibody therapy, chemotherapy and radiotherapy.
A patient who exhibited resistance to postoperative adjuvant
therapy after mastectomy was selected from HER2‑positive
breast cancer, and this patient had the grade of T4bN2a M0,
Stage IIIB. The patient samples, blood serum and cancer
tissue, were analyzed by metabolome and immunostaining
technique, respectively. The characteristics of peripheral
blood serum and solid tumor were investigated, aiming to find
new serum biomarker(s) using the metabolomics technique. A
correlation between the appearance of HER2‑positive cancer
tissue and serum concentration of the sphingomyelin family
was found. In addition, HER2‑positive tumor tissue in both theprimary and recurrent cancer express the sphingomyelinase.
These results suggest that sphingomyelins from this cancer
tissue leads to therapy resistance, induction of invasion andstrong proliferation.

Correspondence to: Dr Satoru Monzen, Department of Radiation
Science, Hirosaki University Graduate School of Health Sciences,
66‑1 Hon‑cho, Hirosaki, Aomori 036‑8564, JapanE‑mail: monzens@hirosaki‑u.ac.jp

Key wards: HER2-positive breast cancer, postoperative adjuvant
therapy, resistant, metabolomics analysis, sphingomyeline

Introduction
Breast cancer is the second most common cancer worldwide withregard to incidence. Two million new cases were reached in 2018,
a number that is continually on the increase (1). Invasive ductal
carcinoma (IDC) is most common type, constituting ~70‑80%
of all breast cancer diagnoses (2). Abnormal cancer cells thatform in the milk ducts spread beyond the ducts into other partsof the breast tissue (2). In particular, tumors with overexpressedor amplified human epidermal growth factor receptor 2 (HER2)
show strong proliferation, migration and invasion. Early treat‑ment is necessary, most commonly in the form of chemotherapy
and surgery (3). Recently, HER2‑directed targeted therapy using
monoclonal antibodies, such as Trastuzumab and Pertuzumab,
has been established as standard of care. Trastuzumab acts
directly against the extracellular domain of the HER2 receptor
and prevents ligand‑independent HER2 signaling, downregu‑
lates HER2 expression and reduces the more active p95‑HER2
form of HER2. Pertuzumab binds to the extracellular domain II
of HER2 and inhibits ligand‑dependent HER2‑HER3 dimeriza‑tion. These monoclonal antibody therapies are recommended
for use together with radiotherapy and/or chemotherapy (4‑8).
However, some tumors are resistant to treatment by antibody
therapy, chemotherapy and radiotherapy. The mechanisms of theresistance are not known.
In order to gain more insight into the resistance mecha‑
nisms, a metabolome analysis of serum collected repeatedly
from a HER2‑positive breast cancer patient who showed
resistance to postoperative adjuvant therapy (PAT) was
performed. The focus was on low‑molecular‑mass metabolites
in blood because they, better than other ‘omic’ profiles, reflect
the patients' biochemical status or physiopathological condi‑
tion. Alterations at the metabolomic level not only reflect thealterations at the genomics and proteomics levels, but also are
influenced by environmental factors (9).
In recent years, metabolomic studies have been successfully
used to identify biomarkers and altered metabolic pathways in

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MONZEN et al: RESISTANCE BIOMARKER IN HER2+ BREAST CANCER

various cancer systems, including gastric, brain, breast, andlung cancer (10‑13). In particular, a metabolomics approach
was applied to identify biomarkers potentially associated withpathological complete response to trastuzumab‑paclitaxel
neoadjuvant therapy in HER2‑positive breast cancer patients.
Consequently, good responders showed higher levels of sper‑
midine and lower amounts of tryptophan compared to the poor
responders (14).
Patients and methods
Study subject and ethics approval. The PAT‑resistant patient
was selected from HER2‑positive breast cancer patients (all>40 years) who were treated by mastectomy at the Mutsu
General Hospital (Aomori, Japan) between February, 2017 andOctober, 2019. During this research period, a PAT‑resistantcase was found and further analysis was performed. The
characteristic treatment flow and investigational points
are summarized in Fig. 1A. The diagnosis was ductal
adenocarcinoma in primary and solid tubular et scirrhous
carcinoma in recurrent, T4bN2a M0, Stage IIIB. The patient
was treated using Fluorouracil + Epirubicin Hydrochloride +Cyclophosphamide Hydrate (PAT1; day 18‑78), and then with
Trastuzumab + Paclitaxel (PAT2; day 102‑466) (Fig. 1A).
External radiotherapy (photon beam, 50 Gy per fraction,
25 fractions) was carried out on day 494‑529 (PAT3). After
surgery for removal of recurrent cancer tissue, the patient was
treated using Trastuzumab + Pertuzumab + Docetaxel (PAT4;
from day 669).
The study was approved by the Committee of Medical
Ethics of the Hirosaki University Graduate School of Health
Sciences (Hirosaki, Japan; no. 2016‑051) and Mutsu General
Hospital (Mutsu, Japan; no. 000‑282) to ensure the welfare andprivacy of the donors. Following a detailed verbal explanation
regarding the content of this study, a written informed consent
was obtained.
Follow up by blood test. To observe the patient's condition
during the treatment, counts of white blood cells (WBC), redblood cells (RBC) and platelets (Plt) were recorded and thelevel of hemoglobin (Hg) was measured by routine hemato‑
logical techniques. At the same time, the concentration of
C‑reactive protein (CRP), carcinoembryonic antigen (CEA),
cancer antigen 15‑3 (CA15‑3) and breast cancer antigen 225(BCA225) were also quantified.
Collection of peripheral blood (PB) serum for metabolomics
analysis. PB was collected directly into serum separation
tubes (BD Biosciences). The serum was separated and storedat ‑80˚C.
Mass spectrometry for metabolomics. Serum metabolites
were measured as part of a targeted metabolomics panel
using the AbsoluteIDQ P180 kit (Biocrates, Inc.). The kit is afully automated assay based on phenylisothiocyanate (PITC)
derivatization of the target analytes using internal standards
for quantitation. Human serum sample preparation was carriedout according to the manufacturer's protocol. Briefly, 10 µl of
serum was transferred to the first 96‑well plate and dried undera nitrogen stream. Thereafter, 50 µl of a 5% PITC solution wasadded to derivatize amino acids and biogenic amines. Afterincubation, the filter spots were dried again before the metabo‑lites were extracted using 5 mM ammonium acetate in methanol(300 µl) into the second 96‑well plate for analysis after further
dilution using the Biocrates MS running solvent A.
Acylcarnitines, lyso‑phosphatidylcholines with acyl
residue, phosphatidylcholine with diacyl residue sum (PC aa),and phosphatidylcholine with acyl‑alkyl residue sum (PC ae),sphingomyelins, and the sum of hexoses were analyzed by theflow injection analysis (FIA) method in positive ion mode using
the liquid chromatography‑mass spectrometry (LC‑MS/MS).
The LC‑MS/MS analysis was carried out by an HPLC system(ExionLC™ AD, AB Sciex) coupled to a QTRAP6500+ triple
quadruple ion trap hybrid mass spectrometer system (ABSciex) in electrospray ionization (ESI) mode. The full list of
metabolites and their abbreviations are presented in Table I.
The metabolites were identified and quantified with multiple
reaction monitoring (MRM) with individual transition andparameters of declustering potential (DP) and collision energy(CE) as listed in Table S1. Flow rate settings for FIA were as
follows: 0.03 ml/min in 1.6 min; 0.03 to 0.2 ml/min in 0.8 min;
0.2 ml/min in 0.4 min; 0.2 to 0.03 ml/min in 0.2 min. MS
settings for FIA mode were as follows: Curtain gas, 45; ion
spray voltage, 5,500 V; temperature, 175˚C; ion source gas 1,
40 psi; ion source gas 2, 50 psi; CAD gas, 6 psi; entrance poten‑
tial, 10 V; collision cell exit potential, 15 V. Twenty microlitersof the sample extract were used in the FIA. Quantification was
carried out using internal standards and a calibration curve.
Amino acids and biogenic amines were analyzed via
LC‑MS/MS in positive ion mode. Then, 10 µl of the sample
extract were injected onto an HPLC C18 column (Zorbax
Eclipse XDB‑C18 column, 3x100 mm, 3.5 µm; Agilent)with a guard column (Zorbax SB‑C18, 3x100 mm, 1.85 µm;
Agilent) at 40˚C using a 10‑min solvent gradient employing
0.2% formic acid in water (solvent A) and 0.2% formic acid in
acetonitrile (solvent B). Additional LC settings for LC‑MS/MS
were as follows: 0% B in 0.5 min; 0‑95% B in 5 min; 95% B
in 1 min; 95‑0% B in 0.5 min; 0% B in 2.5 min at a flowrate of 0.5 ml/min. MS settings for LC‑MS/MS mode were as
follows: Curtain gas, 45; ion spray voltage, 5,500 V; tempera‑ture, 500˚C; ion source gas 1, 40 psi; ion source gas 2, 50 psi;
CAD gas, 6 psi; entrance potential, 10 V; collision cell exit
potential, 15 V. All metabolites were identified and quantified
using isotopically‑labeled internal standards and MRM as
optimized and provided by Biocrates Inc.
Immunological analysis in HER2‑positive cells in cancer
tissue sections. Paraffin‑embedded sections of the removed
cancer tissues were deparaffinized with xylene and ethanol,
washed in D‑PBS (‑) and treated with 3% hydrogen peroxidefor 5 min. The slides were treated with 10  mM citrate buffer(pH 6.0) and incubated for 10 min at 121˚C using an autoclave.
Then, the slides were washed in TBS buffer (25 mM Tris‑HCland 150 mM NaCl, pH 7.2). To perform blocking, the slides
were treated with the blocking solution (5% normal goat serum
in TBS buffer) at room temperature for 30 min. Next, theslides were incubated with the blocking solution containing a
primary rabbit monoclonal antibody directed against human
HER2/ERBB2 (no. 4290; Cell Signaling Technology Inc.) at
a 1:1,000 dilution at room temperature for 60 min. The slides

MOLECULAR AND CLINICAL ONCOLOGY 13: 70, 2020

3

Figure 1. Treatment flow and HER2‑expression. (A) Treatment flow of the studied HER2‑expressing breast cancer patient. (B) Immunological staining ofbreast cancer tissue. Primary cancer tissue was removed by surgery immediately after diagnosis (day 0). The 1st recurrent tumor tissue was removed on
day 656 and 2nd recurrent tumor tissue was detected on day 911 (indicated by inverse triangles). The indicated days (650, i), (669, ii) and (809, iii) denote thecollection of blood serum samples for metabolomic analysis. The administrated PATs were [1]: Fluorouracil + Epirubicin Hydrochloride + Cyclophosphamide Hydrate, [2]: Trastuzumab + Paclitaxel, [3]: External X‑beam therapy (50 Gy in 25 daily fractions), [4]: Trastuzumab + Pertuzumab + Docetaxel. PAT,
postoperative adjuvant therapy. The expression of HER2 antigen in normal and tumor milk ductal tissues was analyzed in tissue samples removed on day 0.
The depth regions of normal and tumor tissue were also analyzed.

were washed three times with TBS buffer and incubated atroom temperature for 60 min with an anti‑rabbit IgG horse‑radish peroxidase (HRP)‑linked antibody (no. 7074, Cell
Signaling Technology) prepared in the blocking solution at a
1:2,000 dilution. The slides were washed three times with TBS
buffer at room temperature for 5 min. Signals were visualizedusing 3,3'‑diaminobenzidine (DAB) (Sigma‑Aldrich). The
slides were washed in water and nuclei were stained using
hematoxylin solution. Finally, the slides were dehydrated
with ethanol and xylene, and mounted using a hydrophobic
mounting agent. Fluorescent immunological analysis was
performed as in our previous study (15). Mouse monoclonal
antibody directed against human nSMase2 (sc‑166637; Santa
Cruz Biotechnology, Inc.) (1:100 dilution) and rabbit mono‑clonal HER2/ERBB2 antibody (1:1,000 dilution) describedabove was used as a primary antibody. Anti‑rabbit IgG Alexa
Fluor 488‑conjugated antibody (no. 4412, Cell Signaling
Technology) and Anti‑mouse IgG Alexa Fluor 647‑conjugatedantibody (no. 4410, Cell Signaling Technology) were used as
secondary antibodies. The cell nuclear staining for fluores‑
cence analysis DAPI (no. 4083; Cell Signaling Technology)
was used. Fluorescence signals were detected using a fluores‑
cence microscope (BZ‑X700; Keyence).

the tumor was removed by surgery (day 0, Fig. 1A). Threemonths after completing PAT3 (day 606), locally recurrent
cancer lesion in right breast within radiation field was detected
and removed by surgery on day 656. A second locally recur‑
rent cancer lesion and metastatic lymph node tumor wereobserved on day 911. According to pathological analysis, theremoved primary tumor tissue (day 0) showed a distinctly
higher expression of HER2 as compared to corresponding
areas of normal tissue (Fig. 1B). A transient increase in WBC(days 1 and 657) and Plt (days 7‑102 and 669) after surgery wasobserved (Fig. 2A and B). On the other hand, the number andconcentration of RBC was stable (Fig. 2C and D). The inflam‑mation marker CRP responded similarly to WBC and Plt(Fig. 2E), whereas the level of CEA remained stable (Fig. 2F).
The levels of CA15‑3 and BCA225 gradually increased during
the observation period however these markers were within thenormal range (Fig. 2G and H).

Results

Repeated tumor recurrence during PAT. After the first surgery,4 cycles of PAT were performed (Fig. 1A). According to patho‑
logical analysis, the staging T1N1M0 (Stage IIA) at the firstdiagnostic stage (before day 0) changed to rT4bN2aM0 when therecurrent tumor was detected in the same original region andwith a part invading into the greater pectoral muscle as well as
presenting as lymph node metastasis (~day 650).

Patient description. Following diagnosis of right breast adeno‑carcinoma with the expression of HER2 as primary cancer,

Metabolomic analysis in PB serum. None of the four PAT
cycles showed effectiveness. To investigate whether specific

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MONZEN et al: RESISTANCE BIOMARKER IN HER2+ BREAST CANCER

Table I. The classification of metabolites in this study using the Biocrates AbsoluteIDQ™ kit.
Metabolite classAmino acid biogenic amine

Carnitine acylcarnitine
Hydroxy‑ and dicarboxyacylcarnitines
Sphingomyeline, hydroxysphingomyelins

Diacyl phosphatidylcholine

Acyl‑alkyl phosphatidylcholine

Lysophosphatidylcholine

Sugar
Total

No. of analytes

Analyte name42	Ala, Arg, Asn, Asp, Cit, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Orn,Phe, Pro, Ser, Thr, Trp, Tyr, Val, Ac‑Orn, ADMA, alpha‑AAA,
c4‑OH‑Pro, Carnosine, Creatinine, DOPA, Dopamine, Histamine,
Kynurenine, Met‑SO, Nitro‑Tyr, PEA, Putrescine, SDMA,
Serotonin, Spermidine, Spermine, t4‑OH‑Pro, Taurine, total DMA
26	C0, C2, C3, C3:1, C4, C4:1, C5, C5:1, C6 (C4:1‑DC), C6:1, C8,
C9, C10, C10:1, C10:2, C12, C12:1, C14, C14:1, C14:2, C16,
C16:1, C16:2, C18, C18:1, C18:2
14	C3‑DC (C4‑OH), C3‑OH, C5‑DC (C6‑OH), C5‑M‑DC, C5‑OH(C3‑DC‑M), C5:1‑DC, C7‑DC, C12‑DC, C14:1‑OH, C14:2‑OH,
C16‑OH, C16:1‑OH, C16:2‑OH, C18:1‑OH
15	SM (OH) C14:1, SM (OH) C16:1, SM (OH) C22:1, SM (OH)
C22:2, SM (OH) C24:1, SM C16:0, SM C16:1, SM C18:0, SM
C18:1, SM C20:2, SM C22:3, SM C24:0, SM C24:1, SM C26:0,
SM C26:1
38	PC aa C24:0, PC aa C26:0, PC aa C28:1, PC aa C30:0, PC aa
C30:2, PC aa C32:0, PC aa C32:1, PC aa C32:2, PC aa C32:3, PC
aa C34:1, PC aa C34:2, PC aa C34:3, PC aa C34:4, PC aa C36:0,
PC aa C36:1, PC aa C36:2, PC aa C36:3, PC aa C36:4, PC aa
C36:5, PC aa C36:6, PC aa C38:0, PC aa C38:1, PC aa C38:3, PC
aa C38:4, PC aa C38:5, PC aa C38:6, PC aa C40:1, PC aa C40:2,
PC aa C40:3, PC aa C40:4, PC aa C40:5, PC aa C40:6, PC aa C42:0,
PC aa C42:1, PC aa C42:2, PC aa C42:4, PC aa C42:5, PC aa C42:6
38	PC ae C30:0, PC ae C30:1, PC ae C30:2, PC ae C32:1, PC ae
C32:2, PC ae C34:0, PC ae C34:1, PC ae C34:2, PC ae C34:3, PC
ae C36:0, PC ae C36:1, PC ae C36:2, PC ae C36:3, PC ae C36:4,
PC ae C36:5, PC ae C38:0, PC ae C38:1, PC ae C38:2, PC ae
C38:3, PC ae C38:4, PC ae C38:5, PC ae C38:6, PC ae C40:1, PC
ae C40:2, PC ae C40:3, PC ae C40:4, PC ae C40:5, PC ae C40:6,
PC ae C42:0, PC ae C42:1, PC ae C42:2, PC ae C42:3, PC ae C42:4,
PC ae C42:5, PC ae C44:3, PC ae C44:4, PC ae C44:5, PC ae C44:6
14	lysoPC a C14:0, lysoPC a C16:0, lysoPC a C16:1, lysoPC a C17:0,
lysoPC a C18:0, lysoPC a C18:1, lysoPC a C18:2, lysoPC a C20:3,
lysoPC a C20:4, lysoPC a C24:0, lysoPC a C26:0, lysoPC a C26:1,
lysoPC a C28:0, lysoPC a C28:1
1
H1
188

Cx:y (x, number of carbons in the fatty acid side chain; y, number of double bonds in the fatty acid side chain); DC, decarboxyl; OH, hydroxyl;M, methyl; PC, phosphatidylcholine; aa, acyl‑acyl; ae, acyl‑alkyl; a, lyso; SM, sphingomyelin.

biomarker(s) were released from cancer tissues to circulating
body fluids, PB serum was collected at the time before
surgery for 1st recurrent tissue (day 650, Fig. 1‑i), after
13 days from surgery (day 669, Fig. 1ii) and the day whenthe 2nd recurrent cancer was diagnosed (day 809, Fig. 1‑iii),
and analyzed by mass spectrometry for wide‑targeted
metabolomic markers of response. The concentration of
188 molecules was analyzed (Table I), and, of these, 40
upregulated molecules and 28 downregulated molecules
were observed on day 669 (Fig. 1A‑ii) as compared today 650 (Fig. 1A‑i) (Fig. 3A and B) by merely calculating themolecules in Table I. In addition, 23 molecules among the
40 upregulated molecules (Fig. 1A‑ii) were downregulated on
day 809 (Fig. 1A‑iii) in comparison to day 669 (Fig. 1A‑ii).
On the other hand, 23 molecules of the 28 downregulated
molecules on day 669 (as compared to day 650) wereupregulated on day 809 (as compared to day 669). Thesemolecules that were up‑to‑down‑ and down‑to‑up‑regulated
contained many lipids of three classes: Acyl‑alkyl phospha‑
tidylcholine (6 molecules), sphingomyeline (4 molecules) andlysophosphatidylcholine (lysoPC) (7 molecules) (Fig. 3C).
Interestingly, the concentrations of molecules in these threeMOLECULAR AND CLINICAL ONCOLOGY 13: 70, 2020

5

Figure 2. Peripheral blood cell counts, haemoglobin, and biochemical markers during the course of therapy. Concentrations of (A) white blood cell (WBC),(B) platelet (Plt), (C) red blood cell (RBC), (D) level of hemoglobine (Hb), (E) C‑reactive protein (CRP) inflammation marker, and (F, G and H) breast
cancer‑specific markers carcinoembryonic antigen (CEA), cancer antigen 15‑3 (CA15‑3) and breast cancer antigen 225 (BCA225), respectively. The normal
range of WBC, Plt, RBC, Hb, CRP, CEA, CA15‑3 and BCA225 were: 4.5‑11.0x106/ml, 1.5‑4.5x105/ml, 4.2‑5.4x106/ml, 12.5‑15.5 g/dl, <3.0 mg/ml, <5.0 ng/ml,
30 U/ml and <160 U/ml, respectively.

classes showed fluctuations which correlated with the detec‑tion of cancer recurrence (Fig. 3C). LysoPC is produced frompartial hydrolysis of phosphatidylcholines, and is regarded as
an inflammation‑resolution lipid mediator (16). In this casestudy, half of the 14 lysoPC species measured were increased
after surgery and decreased when cancer recurred.
I m m u n o e x p re s s i o n a n a l y s i s i n c a n c e r t i s s u e.
Phosphatidylcholines and sphingomyelins are known
as components of cell membranes, especially in extra‑
cellular vesicles which are associated with cell‑to‑cellcommunication (17‑19). Therefore, we focused on neutral
sphingomyelinase‑2 (nSMase), which is one of the importantfactors for exocytosis or endocytosis. In primary cancer tissue
and 1st recurrent cancer tissue, a higher expression of Her2
protein was observed (Fig. 4). However, the lowest accumula‑tion of intracellular nSMase in recurrent tumor was shown incomparison to normal or primary tumor. This means that theexocytosis or endocytosis occurs for release as extracellular
vesicles (EVs).
Discussion
In the present study, we focused on a single HER2‑positive
breast cancer patient with PAT resistance to identify the char‑acteristics of serum metabolites using metabolome analysis.
The applied PATs were not effective in their anti‑tumorigenic,anti‑invasion and anti‑lymph node metastatic action. In
addition, the biochemical markers in peripheral blood did
not respond to the PATs in a strong manner. It is known that

~60% of patients with HER2‑positive breast cancer develop
resistance to trastuzumab, partially due to loss of expressionof HER2 extracellular domain on their tumor cells (20). In
clinical sites, this is sometimes diagnosed as a resistant frac‑tion to administration of chemotherapeutic drugs (i.e., taxans)
and/or external photon radiotherapy in HER2‑positive cancer
patients. However, the essential cause or the key factor driving
this resistance remains to be determined. Previous findings haveshown that the PI3K/Akt/mTOR pathway, STAT3‑survivin
signaling and expression of mitogen‑activated protein kinase
phosphatase 1 produce for an environment that promotes
resistance (21‑23). Therefore, there may be different subpopu‑
lations of cancer cells that are responsive or non‑responsive tocytotoxic drugs or ionizing radiation in HER2‑positive breast
cancer.
A poor response of traditional breast cancer biomarkers inserum (CEA, CA15‑3 and BCA225) was observed in the bloodof the studied patient (Fig. 2) and they were of no value forpredicting the recurrence of cancer until the pathological test
was performed and diagnosed with correctly (day 645‑650).
Therefore, the identification of earlier specific biomarkers
of HER2‑positive breast cancer progression and therapeutic
effectiveness is needed. We aimed to identify new serum
biomarker(s) using the metabolomics technique and foundthat the concentration of the sphingomyeline family in serumis inverse‑correlated to the accumulation of nSMase in
HER2‑positive cancer tissue (Figs. 3 and 4). Sphingomyelin
is one of the sphingolipids, an important structural componentof biological membranes and one of the end‑points in thesynthesis of sphingolipids (24). Its hydrolysis releases ceramide

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MONZEN et al: RESISTANCE BIOMARKER IN HER2+ BREAST CANCER

Figure 3. Serum metabolites in blood samples collected on days 650, 669 and 809 (further details in Fig. 1); 1.3‑fold up‑regulated (Up) and 0.7‑fold down‑regulated(Down) molecules were extracted. (A) Overall map of the number of regulated metabolite molecules at different time points. (B) The molecules were classified
into seven components. (C) The detailed molecules in the classes ‘Sphingomyelin’, ‘Acyl‑alkyl phosphatidylcholine’, and ‘LysoPC’ were specified.

and phosphocholine and several stimuli are known to activate
sphingomyelin hydrolysis (25). Ceramide is an important
promoter of apoptosis (18), but additionally, ceramide also
triggers budding of EVs, such as exosomes into multivesicularendosomes (26). It is known that EVs are nanometer‑scaleparticles that are secreted by cells and mediate intercellularcommunication by transferring biomolecules between cells.
Thus, their expression relates to cancer tissue growth or distantmetastasis. We have previously reported that during the active
release of EVs to blood flow after exposure to higher dose ofionizing radiation, the lower accumulation of EV components
in their original tissue cells was observed in comparison to theother tissues with non‑active release of EVs (27,28). Our casedata showed that the concentration of nSMase co‑expressedwith HER2 in 1st recurrent cancer tissue, where the levels ofboth are lower than in primary cancer tissue (Fig. 4), and a

MOLECULAR AND CLINICAL ONCOLOGY 13: 70, 2020

7

Figure 4. Identification of hematoxylin and eosin staining tissue (H&E), HER2‑positive tissue (green) and nSMase‑positive tissue (red) in surgically removed
breast cancer tissues. DAPI (blue) staining that is able to identify the cell nuclear and merged images are also shown.

higher concentration of sphingomyelin family in serum was
detected when the 2nd recurrent cancer tissue was diagnosed.
These responses suggested that recurrent cancer tissues moreeasily release EVs to peripheral blood serum and promote
cancer progression and expansion. Thus, one reported mecha‑
nism is the transfer of miRNAs via exosomes from cancer cells
to microenvironmental cells, promoting angiogenesis (28,29).
It is possible that full resistance to PAT could be connectedto sphingomyelines, which may serve as a serum marker. The
gene sphingomyeline phosphodiesterase 3 (SMPD3) whichcodes for nSMase was shown to act as a tumor suppressor genein hepatocellular carcinoma, supported by overexpression andknockdown experiments (30). Of note, a recent study showedthat high levels of sphingomyelin synthase 2 (SGMS2), which
controls the synthesis of sphingomyelins from ceramide andthereby disrupts the ceramide‑associated apoptosis pathway,
is correlated with breast cancer aggressiveness and metas‑
tasis (31).
Additionally, we saw a clear opposite pattern for diacyl andacyl‑alkyl phosphatidylcholines, where there was a decreaseat 30 days after surgery, and then an increase, versus lysoPCs,
which was initially increased and then decreased. Histologicaldamage and infection caused by surgery induce inflammatory
biological responses which are evoked as a result of exog‑
enous and endogenous mediators secreted not only locally
but also systemically. Of note is that increased levels of the
enzyme lysoPC acyltransferase 1 (LPCAT1), controlling theconversion of lysoPC to phosphatidylcholine, were linked
to poor prognosis and adverse features such as estrogen andprogesterone receptor negativity, amplification of HER2

and MYC and deletions of PTEN and CDKNA2 in breast
cancer (32,33). Kim et al also reported a similar statement that
lipid profiles, such as sphingomyelin and phosphatidylcholine,
correlated with various clinicopathological characteristics of
HER2‑positive breast cancer (34).
Although the PAT resistance could not be clearly deci‑
phered, our results provide novel information of a parallel
expression intensity of HER2 and nSMase, which possibly
is connected to cell communication by EVs. Recently, altera‑tion of gene expression by EV transportation was reported toinduce trastuzumab‑resistant HER2‑positive metastatic breast
cancer (35). Clearly, additional, detailed analysis is required
on a larger number of patients.
In conclusion, complete therapeutic‑resistant cancer tissue
connected to reduced expression of nSMase in HER2‑positive
breast cancer are strongly co‑expressed with the sphingo‑
myelin family which can be detected at the peripheral blood
serum level.
Acknowledgements
The authors are grateful to Miyu Miyazaki at the Center
for Scientific Equipment Management, Hirosaki University
Graduate School of Medicine, for assistance with LC‑MS/MS
analysis.
Funding
This study was supported by ‘JSPS KAKENHI, Grant‑in‑Aid
for Scientific Research (C) (General) (project no. 16K10339,

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MONZEN et al: RESISTANCE BIOMARKER IN HER2+ BREAST CANCER

Satoru Monzen)’, ‘JSPS KAKENHI, Fund for the Promotionof Joint International Research (Fostering Joint International
Research) (project no. 17KK0181, Satoru Monzen)’, and‘Interdisciplinary Collaborative Research Grant for Young
Scientists, Hirosaki University’.
Availability of data and materials
All data generated or analysed during the present study areincluded in this published article.
Authors' contributions
SM and YM designed the study and prepared the manuscript
draft and substantively participated in revising the manu‑
script. SM, YT and MC conceived the study, and contributedby analyzing the patient's data and revised the manuscript.
AW and LL supervised the study and critically reviewed
the manuscript, and gave final approval of the version to be
published. All authors read and approved the final manu‑
script.
Ethics approval and consent to participate
The study was approved by the Committee of Medical Ethicsof the Hirosaki University Graduate School of Health Sciences,(no. 2016‑051) and Mutsu General Hospital (no. 000‑282) toensure the welfare and privacy of the donors. Following adetailed verbal explanation regarding the content of this study,
a written informed consent was obtained.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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