Tamoxifen-resistant breast cancer cells exhibit reactivity with Wisteria floribunda agglutinin.

Glycosylation is one of the most important post-translational modifications of cell surface proteins involved in the proliferation, metastasis and treatment resistance of cancer cells. However, little is known about the role of glycosylation as the mechanism of breast cancer cell resistance to endocrine therapy. Herein, we aimed to identify the glycan profiles of tamoxifen-resistant human breast cancer cells, and their potential as predictive biomarkers for endocrine therapy. We established tamoxifen-resistant cells from estrogen receptor-positive human breast cancer cell lines, and their membrane-associated proteins were subjected to lectin microarray analysis. To confirm differential lectin binding to cellular glycoproteins, we performed lectin blotting analyses after electrophoretic separation of the glycoproteins. Mass spectrometry of the tryptic peptides of the lectin-bound glycoproteins was further conducted to identify glycoproteins binding to the above lectins. Finally, expression of the glycans that were recognized by a lectin was investigated using clinical samples from patients who received tamoxifen treatment after curative surgery. Lectin microarray analysis revealed that the membrane fractions of tamoxifen-resistant breast cancer cells showed increased binding to Wisteria floribunda agglutinin (WFA) compared to tamoxifen-sensitive cells. Glycoproteins seemed to be responsible for the differential WFA binding and the results of mass spectrometry revealed several membrane glycoproteins, such as CD166 and integrin beta-1, as candidates contributing to increased WFA binding. In clinical samples, strong WFA staining was more frequently observed in patients who had developed distant metastasis during tamoxifen treatment compared with non-relapsed patients. Therefore, glycans recognized by WFA are potentially useful as predictive markers to identify the tamoxifen-resistant and relapse-prone subset of estrogen receptor-positive breast cancer patients.

Introduction

Breast cancer is one of the most prevalent diseases in women in Japan, where the incidence and mortality rate has been increasing annually [1]. Based on hormone-receptor (HR) and human epidermal growth factor receptor-2 (HER2) status, breast cancer is classified as either HR-positive HER2-negative, HR-positive HER2-positive, HER2 type, or triple-negative breast cancer [2]. For patients with HR-positive breast cancer, an adjuvant endocrine therapy (ET) will be administered for five to ten years following curative surgery [3, 4]. ET includes selective estrogen receptor modulators, selective estrogen receptor downregulators and aromatase inhibitors, and treatment is based on the patient’s age and side effects. Although early detection techniques, therapeutic regimens and understanding of the molecular basis of breast cancer biology has been advancing, nearly 30% of all patients with early stage breast cancer have recurrence, and most cases are metastatic [5]. De novo and acquired resistance to these therapies is one of the major causes of breast cancer mortality [6] where several molecules may contribute to the acquisition of ET resistance, such as those in the PI3K/mTOR and RAS/RAF/MEK/ERK pathways [7, 8]. Epigenetic alterations can also facilitate the escape of tumor cells from ETs [9]. The combination of hormone therapy with CDK4/6 inhibitors and selective inhibitors of PI3K and mTOR can increase progression-free survival in clinical practice [10]. Although treatments to overcome recurrence and resistance of breast cancer are constantly being developed, the mechanism of resistance is still difficult to understand. A new approach that does not employ the conventional gene and protein expression analysis might be required to uncover these points of resistance.

Glycosylation is a major post-translational modification of proteins through the sequential actions of glycosyltransferases [11, 12]. Alteration of glycan is known to be associated with carcinogenesis, malignant progression and metastasis [13]. Glycosylation changes have also been implicated in drug resistance of cancer. Multidrug resistance in cancer is mainly caused by high expression of P-glycoprotein [14]. For example, profiling of a cisplatin-resistant ovarian cancer cell line with a lectin array and a gene expression array revealed higher expression of core fucose, poly N-acetyllactosamine, and high mannose structures compared with the parental cells [15]. Furthermore, resistance to paclitaxel and other chemotherapeutic agents are associated with altered glycans in breast cancer [16, 17]. However, there have been no studies focused on the role of glycosylation in endocrine resistance in HR-positive breast cancer cells. Here, we report the presence of glycans recognized by Wisteria floribunda agglutinin (WFA) in tamoxifen (TAM) resistant HR-positive breast cancer cells and surgical specimens from patients who exhibited early recurrence after TAM treatment.

Materials and methods

Establishment of TAM-resistant cells

The human breast cancer cell lines T47D and ZR75-1 were purchased from the American Type Culture Collection. They were cultured in Gibco Dulbecco’s Modified Eagle’s Medium (Thermo Fisher Scientific) with 10% fetal bovine serum and 100 U/mL penicillin/streptomycin in a humidified incubator with 5% CO2 at 37°C.

TAM (4-hydroxytamoxifen, H7904) was purchased from Sigma-Aldrich and TAM-resistant cells (T47D-TAMR and ZR75-1-TAMR) were established by chronic exposure of the drug-sensitive T47D and ZR75-1 cells to stepwise increases in TAM concentrations, until a resistance concentration was achieved. The resistance to TAM in T47D-TAMR and ZR75-1-TAMR cells was confirmed by a Cell Counting Kit-8 (Dojindo Laboratories) which showed a higher viability of both cell types after TAM treatment compared with the sensitive cells (S1 Fig). The LD50 (50% lethal dose) in T47D and T47D-TAMR cells was 4.0 μM and 6.3 μM, respectively, and in ZR75-1 and ZR75-1-TAMR cells was 9.5 μM and 10.4 μM, respectively.

Membrane protein extraction

Pellets containing 1x107 cells were collected and the membrane protein (hydrophobic fraction) was extracted using CelLyticTM MEM Protein Extraction Kit (Sigma Aldrich) according to the manufacturer’s protocol. The protein concentration of the obtained hydrophobic fraction was quantified with a PierceTM BCA assay kit (Thermo Scientific).

Lectin microarray analysis

Membrane proteins were diluted with phosphate buffered saline (50 μg/mL) and 20 μL aliquots were labelled with 100 μg of Cy3 succinimidyl ester (GE Healthcare) and incubated at room temperature for 1 h in the dark. Excess labeling reagent was removed by spin column (Zeba spin, Thermo Fisher Scientific), and the treated Cy3-labeled samples diluted with probing solution to 500 ng/mL (GlycoTechnica Ltd). Aliquots (100 μL) of the prepared Cy3-labelled samples were applied to the LecChip (GlycoTechnica Ltd) and incubated overnight at 20°C with gentle shaking, to allow for complete formation of the lectin-glycan complex. Then, the lectin microarray was washed three times with the Probing Solution and scanned with a GlycoStationTM Reader 1200 (GlycoTechnica Ltd). Image scanning was performed at the highest net intensity for 45 lectins, around 50,000 arbitrary fluorescence units. Fluorescence intensities were analyzed using GlycoStationTM Signal Capture Ver. 1.5 and GlycoStationTM ToolsPro Suite Ver. 1.5 (GlycoTechnica Ltd). The average intensity of the three spots for each lectin were normalized using the average value of 45 lectins. The normalized average value was compared between the sensitive and resistant breast cancer cell lines.

Lectin blotting

Membrane protein fractions (10 μg) were loaded per lane, separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and blotted on a polyvinylidene difluoride membrane (Millipore). The membrane was blocked with Bullet Blocking One (Nacalai Tesque) and incubated with biotinylated WFA (2.5 μg/mL; for preparation of biotinylated WFA, please refer to “Clinical samples and lectin staining” below) and biotinylated Sambucus nigra agglutinin (SNA) (2.5 μg/mL) (Vector Laboratories) at 4°C overnight. Membranes were subsequently incubated with horseradish peroxidase conjugated streptavidin (1:4000 dilution, Jackson ImmunoResearch) for 1 h at room temperature. The bands were visualized using Clarity Western ECL detection reagent (Bio-Rad Laboratories) and detected by ChemiDoc Touch (Bio-Rad Laboratories).

Preparation of WFA-binding proteins and identification by liquid chromatography/mass spectrometry (LC/MS) followed by database search using Mascot

For lectin precipitation, prepared hydrophobic fractions (50 μg) diluted in Milli-Q water (up to 40 μL) were subjected to immunoprecipitation using biotinylated WFA. The membrane proteins were preheated at 95°C for 5 min and precleared with 20 μL of Dynabeads My One Streptavidin T1 (Dyna-SA) (Veritas) for 2 h at 4°C. Biotinylated WFA was reacted with Dyna-SA in 20 μL of Tris-buffered saline (TBS) containing 1% Triton-X (TBSTx) for 2 h at 4°C. The beads conjugated with WFA were washed with TBSTx, and precleared membrane proteins were added to the beads that continued overnight incubation at 4°C. The supernatant was removed, and the beads were washed with TBSTx. Washed beads were suspended into 40 μL of TBS containing 0.2% SDS and incubated at 95°C for 10 min to elute the precipitated WFA-binding proteins. Supernatants were collected and shaken with 20 μL of Dyna-SA at 4°C for at least 60 min for depletion of biotinylated WFA which was eluted in the elution step. The samples were centrifuged at 13,000 rpm for 1 min at 4°C and the supernatants were collected and used as the WFA binding fraction.

SDS-PAGE was performed using 10% polyacrylamide gel under a constant current of 30 mA for 53 min. The gel was silver stained (AE-1360 EzStain Silver, Atto), and a band around 100 kDa, corresponding to the bands detected by lectin blotting using WFA, was excised. The cut gel was decolorized for 15 min at room temperature using reagents from the Silver Stain MS kit (Fujifilm Wako). After washing the gel five times with MilliQ water (500 μL each time), the gel was twice washed with acetonitrile (200 μL each time) to dehydrate, and then dried. In-gel digestion was carried out as described previously [18].

Peptide mixture was analyzed by LC/MS using a nanoLC (Ultimate 3000, Thermo Scientific) and Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific). Tandem MS spectra were acquired in a data-dependent manner, where fragmentation was performed by the high-energy collision-induced dissociation method (normalized collision energy = 35). Raw data was converted to an mgf file and applied to a database search using Mascot (Ver. 2.6.2, Matrix Science) and the UniprotKB protein sequence database (42,431 entries). Search conditions were as follows: maximum miss cleavage, 2; peptide mass tolerance, 7 ppm; fragment mass tolerance, 0.02 Da; target false discovery rate, 1%; fixed modification, carbamidomethyl (C); and variable modifications, ammonia-loss (N-terminal C), Gln>pyroGlu (N-terminal Q), and oxidation (M).

Clinical samples and lectin staining

Based on lectin microarray analysis, WFA was chosen for lectin staining. We investigated patients with HR-positive breast cancer who underwent curative surgery at our hospital from December 2012 to April 2018, inclusive, and received TAM as adjuvant therapy. There were 43 patients who developed distant metastasis during five-year-TAM treatment and 20 of these patients were randomly selected for lectin staining. As a control, 20 patients who received the same treatment but were free from distant metastasis were also analyzed. Clinicopathological features of the 40 patients are shown in S1 Table.

For lectin staining, WFA was biotinylated using a Biotin Labeling Kit-NH2 (Dojindo). Formalin-fixed and paraffin-embedded surgical specimens were cut into 3 μm-thick slices and transferred to an automated staining apparatus, BenchMark GX (Ventana Medical Systems). Tissues were incubated with 10 μg/mL biotinylated WFA. Then, sections were stained with diaminobenzidine (Ventana Medical Systems) and counterstained with hematoxylin (Ventana Medical Systems). WFA staining in cancer cells was semi-quantitatively evaluated. We defined WFA as being positive when more than 10% of cancer cells showed staining for this lectin in the cytoplasm and/or the membrane. Among WFA-positive tumors, the staining was then defined as weak positive or strong positive, according to intensity. If both strongly and weakly-stained cells were observed, the dominant intensity was selected. Representative images are shown in S2 Fig.

Ethical approval and informed consent

This study was carried out with approval from the Ethics Committee of Juntendo University Hospital (H19-289) and complies with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. All participants were informed that the research policy was available on the homepage of the hospital and that they had the opportunity to opt-out of the study at any time later on, which was approved by the Ethics Committee. The Ethics Committee approved of the opt-out method for the use of specimens and clinical data under the condition that all data were anonymized. Only those participants who had not opted-out from the study were included in the data analysis.

Statistical analysis

Statistical analyses were performed using JMP 14.2 statistical software (SAS Institute, Inc.). Comparisons of mean values between two groups were performed on unpaired data employing the 2-sided t test. A p-value < 0.050 was considered to indicate a statistically significant difference.

Results

Identification of lectins related to TAM resistance in breast cancer

The membrane protein fraction (hydrophobic) was prepared from cell pellets, and analyzed with the lectin microarray. Among the 45 lectins on the lectin microarray (S3 Fig), the relative intensities of WFA and SNA were higher in T47D-TAMR (p = 0.002, p = 0.125, respectively) and ZR75-1-TAMR cells (p < 0.001, p < 0.001, respectively) than in the TAM-sensitive cells (Fig 1). WFA is known to recognize glycan structures containing terminal N-acetylgalactosamine (GalNAc) or terminal galactose (Gal). SNA is known to recognize sialic acid, which is attached to Gal/GalNAc through α2–6 linkage. To verify the results obtained from the lectin microarray, we performed lectin blotting using WFA and SNA lectins. This confirmed the increased binding intensity of WFA in T47D-TAMR and ZR75-1-TAMR cells compared with sensitive cells; however, SNA binding intensity was not different between the TAM-sensitive and resistant cells (Fig 2). These results suggest that altered glycosylation in breast cancer cells, which can be detected by WFA, could be related to resistance to TAM therapy.

Fig 1

Lectin microarray analysis of T47D and ZR75-1 cells and their TAM-resistant derivative T47D-TAMR and ZR75-1-TAMR cells.

The relative intensities of WFA and SNA in (A) T47D (sensitive) and T47D-TAMR (TAM-resistant) cells and (B) ZR75-1 (sensitive) and ZR75-1-TAMR (TAM-resistant) cells, based on normalized average data. Error bars indicate standard deviations. t tests were employed for statistical comparisons.

Fig 2

Lectin blotting analysis of T47D and ZR75-1 cells and their corresponding TAM-resistant cells.

The hydrophobic fractions from sensitive and resistant cells were electrophoretically separated and the binding of WFA and SNA were examined. (A) The results of lectin blotting in T47D and T47D-TAMR cells. (B) The results of lectin blotting in ZR75-1 and ZR75-1-TAMR cells.

Identification of candidate glycoproteins in T47D-TAMR and ZR75-1-TAMR that bind WFA

Using lectin microarrays and lectin blotting, we decided to focus on the WFA-binding molecules implicated in TAM resistance of breast cancer cells. Lectin blotting after SDS-PAGE identified WFA binding molecules. From the migration position, the molecular mass of the major component had an approximate molecular mass of 100 kDa (Fig 2). The hydrophobic fraction of T47D-TAMR and ZR75-1-TAMR cells was subjected to WFA-lectin precipitation and then analyzed by lectin blotting after SDS-PAGE. WFA binding molecules migrated around 100 kDa (Fig 3A). To identify the candidate molecules that bind to WFA, the WFA-bound fraction was separated by SDS-PAGE and the gel was silver stained (Fig 3B). The gel around 100 kDa was excised and subjected to in-gel tryptic digestion. Peptides extracted from the gel were analyzed by LC/MS. Identified proteins are shown in Table 1, which included CD166 and integrin beta-1.

Fig 3

Precipitation of WFA binding glycoproteins in T47D-TAMR and ZR75-1-TAMR cells.

(A) Blotting of WFA binding fractions with WFA. (B) Silver staining of WFA binding fractions. Red rectangles indicate bands around 100 kDa, which were cut out and used for mass spectrometry.

Table 1

Identified candidate proteins in the WFA binding fraction (100 kDa gel) in T47D-TAMR and ZR75-1-TAMR cells.

T47D-TAMR
Number of identified peptides/accession (WFA binding fraction)	Number of identified peptides/accession (negative control)	Protein accession	Gene Name	Protein description	Protein mass (Da)	Length (amino acids)
37	n.i.*	Q14697	GANAB	Neutral alpha-glucosidase AB	107.263	944
37	n.i.*		GANAB	Isoform 2 of Neutral alpha-glucosidase AB	109.825	966
19	n.i.*	P55060	CSE1L	Exportin-2	111.145	971
18	n.i.*	Q13740	ALCAM	CD166 antigen	65.745	583
15	n.i.*	P07900	HSP90AA1	Heat shock protein HSP 90-alpha	85.006	732
13	n.i.*	P13639	EEF2	Elongation factor 2	96.246	858
13	n.i.*	P14625	HSP90B1	Endoplasmin	92.696	803
12	n.i.*	P02538	KRT6A	Keratin, type II cytoskeletal 6A	60.293	564
12	n.i.*	P05556	ITGB1	Integrin beta-1	91.664	798
8	n.i.*	P19367	HK1	Hexokinase-1	103.561	917
8	n.i.*	Q14525	KRT33B	Keratin, type I cuticular Ha3-II	47.325	404
7	n.i.*	P35221	CTNNA1	Catenin alpha-1	100.693	906
7	n.i.*	Q15323	KRT31	Keratin, type I cuticular Ha1	48.633	416
6	n.i.*	P13646	KRT13	Keratin, type I cytoskeletal 13	49.900	458
5	n.i.*	P04792	HSPB1	Heat shock protein beta-1	22.826	205
5	n.i.*	P32004	L1CAM	Neural cell adhesion molecule L1	140.885	1257
5	n.i.*	Q14533	KRT81	Keratin, type II cuticular Hb1	56.832	505
ZR75-1-TAM R
30	n.i.*	Q13740	ALCAM	CD166 antigen	65.745	583
13	n.i.*	P05556	ITGB1	Integrin beta-1	91.664	798
4	n.i.*	O43493	TGOLN2	Trans-Golgi network integral membrane protein 2	50.988	479
3	n.i.*	P19440	GGT1	Glutathione hydrolase 1 proenzyme	61.714	569
3	n.i.*	P36268	GGT2	Inactive glutathione hydrolase 2	62.074	569
3	n.i.*		GGT2	Inactive glutathione hydrolase 2	61.026	559
3	n.i.*		GGT2	Isoform 3 of Inactive glutathione hydrolase 2	62.538	574
3	n.i.*	Q14697	GANAB	Neutral alpha-glucosidase AB	107.263	944
3	n.i.*		GANAB	Isoform 2 of Neutral alpha-glucosidase AB	109.825	966
3	n.i.*	Q9Y639	NPTN	Neuroplastin	44.702	398
3	n.i.*		NPTN	Isoform 1 of Neuroplastin	31.500	282
3	n.i.*		NPTN	Isoform 3 of Neuroplastin	31.044	278
3	n.i.*		NPTN	Isoform 4 of Neuroplastin	38.110	337
3	n.i.*		NPTN	Isoform 5 of Neuroplastin	44.245	394
2	n.i.*	A6NGU5	GGT3P	Putative glutathione hydrolase 3 proenzyme	61.919	568
2	n.i.*	P05090	APOD	Apolipoprotein D	21.547	189
2	n.i.*	P14625	HSP90B1	Endoplasmin	92.696	803

WFA, Wisteria floribunda agglutinin; TAM, tamoxifen; n.i., not identified

WFA staining of clinical samples

We conducted WFA staining of surgical specimens, and the relationship between binding intensity and patient outcomes was examined. The majority of primary tumors were positive for WFA both in the relapsed group (n = 20) and the control group who were free from recurrent disease (n = 20), with 19 tumors (95%) and 17 tumors (85%) showing strong/weak positivity, respectively (Fig 4). Interestingly, strong WFA staining was more frequently observed in the relapsed group, who developed distant metastasis during TAM treatment, compared with those in the control group (Fig 4, p = 0.011). We further conducted WFA staining in metastatic lesions to compare with primary tumors. Among the 20 metastatic patients, a biopsy of the metastatic lesion was performed in five patients and three of these cases were available for WFA staining. In these three cases, there was no difference in WFA expression between primary and metastatic lesions as WFA was already strongly positive in primary tumors and was maintained in the metastatic lesion (S4 Fig).

Fig 4

Histochemical WFA staining of sections from primary breast cancer specimens.

Primary tumors from patients who developed distant metastasis during adjuvant TAM treatment (n = 20) and those who did not develop recurrent disease (n = 20) were analyzed. Strong positive staining (dark grey) was significantly more frequent in relapsed patients (13 patients, 65%) than in the control group (4 patients, 20%; p = 0.011).

Discussion

Despite early detection techniques, therapeutic regimens and an understanding of the molecular basis of breast cancer biology, more than 30% of patients receiving adjuvant ET develop metastatic disease, during or after treatment [19]. It is critical to identify this group of patients and to design new therapeutic strategies suited to this subset.

Although altered cellular glycosylation is known to be associated with cancer progression and metastasis [11], reports on altered glycosylation in cancer cells resistant to anticancer drugs are limited. One important aspect is that glycosylation is known to modulate the function of molecules involved in apoptosis [20]. We recently reported that Mucin 21 confers resistance to apoptosis in an O-glycosylation-dependent manner [21]. Another aspect is that the function of drug transporters is glycosylation-dependent [22-24], with studies showing that different glycans influence the sensitivity to different drugs through a variety of mechanisms. In breast cancer, although the importance of glycosylation as a biomarker has been extensively investigated [25], there have been no previous studies regarding glycan-based analyses in TAM-resistant luminal type breast cancer cells. In the present study, we performed lectin microarray analysis of membrane fractions of newly developed TAM-resistant breast cancer cells. We found increased WFA-binding in TAM-resistant breast cancer cells, compared with TAM-sensitive cells.

Studies investigating the relationships between serum biomarkers and various diseases have reported the usefulness of WFA binding [26-28]. For instance, Fujiyoshi et al. reported elevated serum WFA-positive Mac-2-binding protein levels were a significant risk factor for tumor recurrence in hepatocellular carcinoma [27]. Most of these previous studies have focused on the changes in WFA binding in serum samples; few studies have focused on WFA binding to cell surface molecules [29]. We previously reported that surgical specimens of relapsed triple-negative breast cancer patients showed higher levels of WFA binding than that of non-relapsed groups, using lectin microarrays [29]. In the present study, we identified WFA as a lectin having differential binding between TAM-resistant and sensitive breast cancer cells. In addition, we found strong WFA staining in the relapsed patients treated with TAM.

Recently, WFA was shown to strongly interact with the LacdiNAc structure produced by human cervical carcinoma cells, and 1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2) is responsible for its biosynthesis [30]. Using publicly available Kaplan–Meier plotter mRNA microarray databases [31], we examined the relationship between B3GALNT2 expression and the recurrence-free survival of patients who were given adjuvant TAM treatment. Patients with tumors containing high levels of B3GALNT2 mRNA exhibited significantly shorter recurrence-free periods than those with low levels of mRNA for this enzyme (S5 Fig). If the mRNA levels correspond to the formation of WFA-reactive glycans, then these indirect database results can be considered consistent with the lectin microarray data and lectin staining data we obtained.

Further investigations are necessary to determine the mechanism of how WFA-binding glycans contribute to TAM-resistance. Che et al. reported that WFA-binding glycoproteins promoted cancer stemness via epidermal growth factor receptor signaling in colorectal cancer [32]. A similar mechanism may be responsible for therapeutic resistance in hormone-positive breast cancer. However, such mechanisms should be explored after the identification of the glycoproteins carrying WFA-binding glycans. Asif et al. reported that activated integrin beta-1 is increased in metastatic breast cancer cells [33], and CD166 is also upregulated in TAM-resistant breast cancer cells [34]. Interestingly, in our present study, integrin beta-1 and CD166 were identified as candidates for WFA binding membrane glycoproteins. However, we could not verify whether the amount of these glycoproteins themselves was greater in resistant cells compared with sensitive cells, or whether structural differences in the glycans of these glycoproteins on the TAM-resistant cells made them reactive with WFA.

In clinical samples, strong WFA staining was more frequently observed in the relapsed patients, compared with non-relapsed patients, indicating that this lectin might have the potential to be a predictive marker for de novo resistance to TAM treatment, not just acquired resistance. When comparing WFA staining intensity in metastatic lesions that developed during TAM treatment, there was no difference in the expression level compared with their paired primary lesion, although we assessed only three patients. Further studies with larger samples are needed to confirm this finding. Moreover, to avoid inter- and intra-observational differences and obtain more reproducible results, another more detailed scoring system should be considered in future studies, as suggested by some recent reports [35, 36].

In conclusion, we found that WFA, which is reported to recognize the LacdiNAc structure containing terminal GalNAc residues, showed stronger binding to TAM-resistant breast cancer cells. Moreover, strong WFA staining was observed in patients who developed distant metastasis during TAM treatment. Our results suggest that further investigation is warranted into glycoproteins with WFA-binding glycans on TAM resistant HR-positive breast cancer cells.

Cell proliferation assays in T47D, ZR75-1 and TAM-resistant cells.

Cell viability of T47D, T47D-TAMR, ZR75-1 and ZR75-1-TAMR cells after 72 h of TAM treatment. The LD50 of T47D and T47D-TAMR cells was 4.0 μM and 6.3 μM, respectively, and that of ZR75-1 and ZR75-1-TAMR cells was 9.5 μM and 10.4 μM, respectively. Error bars indicate standard deviations.

(PDF)

Click here for additional data file.

Representative images of HR-positive breast cancer tissues stained with WFA.

WFA staining was assessed as negative, weak positive or strongly positive. Strong staining was observed mainly in the cell membrane and cytoplasm of HR-positive breast cancer tissues.

(PDF)

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Lectin microarray analysis.

Lectin microarray analysis of (A) T47D and T47D-TAMR cells and (B) ZR75-1 and ZR75-1-TAMR cells, showing the relative intensities of 45 lectins in the TAM-sensitive and resistant cells, based on normalized average data. Error bars indicate standard deviations.

(PDF)

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Comparison of WFA staining in primary and metastatic lesions.

WFA staining of primary and metastatic lesions in three patients are shown. Histological type of patient 2 was mucinous carcinoma. Metastatic lesions of patient 1, 2 and 3 are duodenum, lung, and liver, respectively.

(PDF)

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Kaplan-Meier curves of recurrence-free-survival according to B3GALNT2 mRNA expression (n = 178).

The curve shown in red represents recurrence-free-survival of patients with B3GALNT2 mRNA-high tumors, while the black curve shows those with low B3GALNT2 tumors. The data was obtained from publicly available Kaplan–Meier plotter mRNA microarray databases [29].

(PPTX)

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Raw images of Fig 2 and Fig 3.

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Clinicopathological features of patients.

(XLSX)

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13 Jun 2022

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Additional Editor Comments:

I have recommended major revision for your manuscript, based on the comments and observations made by three reviewers.

Although your manuscript presents primary research that contributes to scientific knowledge, it requires additional work because publication criteria 3 and 4 were not completely covered.

Criteria No. 3. Experiments, statistics, and other analyses are performed to a high technical standard and are described in sufficient detail.

The three reviewers are asking for further methodological descriptions. Moreover, I consider that the inclusion of the MS analysis of sensitive cells should be included as control.

In addition, biofinformatic analysis can be performed in order to determine whether the identified proteins contain the N-acetylgalactosamine beta 1 (GalNAc beta 1-3 Gal) modification that is recognized by the WFA lectin.

Criteria No. 4. Conclusions are presented in an appropriate fashion and are supported by the data.

The manuscript does not contain any experimental results that confirm the role of the identified glycoproteins in the TMX resistance mechanism. For this reason, reviewer No. 1 rejected the manuslcript and reviewer No. 3 is proposing a change in the title of the manuscript. Therefore, authors have to decide if they go deeper in looking for the participation of the glycoproteins identified in the resistance mechanisms or discard this asseveration from the title.

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Reviewers' comments:

Reviewer's Responses to Questions

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: I Don't Know

Reviewer #3: No

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes

**********

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The topic on glycans for drug resistant in breast cancer treamtent is interesting. However, the data

current can not support the finding and conculsions.

1. Glycomics has been studied in other reports, where the modification sites and glycan structures

can be clearly identified. The authors carried out mass spectrometry analysis but did not give out any

meaningful proteomic data (probably only identify some protein sequences)

2.Lectin array can only give the subtype of glycoprotein expression and it should be integrated with

protein expression (WB) to see the glycosylation change.

3. Clinical sample analysis is not very related cell-based study, which need more data to support (e.g. quantitative glycoproteomics on the clinical samples).

Reviewer #2: Line 94: resistant cell lines are described as if they had been developed in the laboratory itself and this is not the case, they were acquired from a commercial company.

Line 100: the term "parental cells" does not apply, although it is the same cell line with acquired resistance they do not come from the same cell passage

Line 256: the proteins identified in the WFA-binding fraction in TAM-cells are mentioned, nevertheless the same analysis should be carried out in the cells not TAM to be able to conclude with greater support the results

Line 264: It is not clear whether the control group refers to patients treated with tamoxifen who did not develop metastases or rather who did not relapse.

Line 307: is incorrectly worded, lectin binding to glycoproteins does not contribute to resistance, but rather could function as a potential biomarker or prognostic factor.

Reviewer #3: In the methods section, the identification of lectin-bound proteins should be mentioned in a single section.

In the methods section, the quantification of the biotinylated lectin reaction in tissue from cancer patients is deficient. It is suggested to explain in detail. Consider published methods to assess different levels of positivity.

1) Diagn Pathol. 2014 Nov 29;9:221.doi: 10.1186/s13000-014-0221-9.Different approaches for interpretation and reporting of immunohistochemistry analysis results in the bone tissue - a review. Nickolay Fedchenko 1 2, Janin Reifenrath 3. DOI: 10.1186/s13000-014-0221-9

2) Pathol Res Pract. 2015 Dec;211(12):973-81.doi: 10.1016/j.prp.2015.10.002. Epub 2015 Nov 6. Integrins and haptoglobin: Molecules overexpressed in ovarian cancer. Julio César Villegas-Pineda 1, Olga Lilia Garibay-Cerdenares 2, Verónica Ivonne Hernández-Ramírez 3, Dolores Gallardo-Rincón 4, David Cantú de León 5, María Delia Pérez-Montiel-Gómez 6, Patricia Talamás-Rohana 7

3) Cancer Cell Int. 2022; 22: 6. doi: 10.1186/s12935-021-02425-6 PHD finger protein 20-like protein 1 (PHF20L1) in ovarian cancer: from its overexpression in tissue to its upregulation by the ascites microenvironment. Dulce Rosario Alberto-Aguilar,1 Verónica Ivonne Hernández-Ramírez,1 Juan Carlos Osorio-Trujillo,1 Dolores Gallardo-Rincón,2 Alfredo Toledo-Leyva,2 and Patricia Talamás-Rohana1

The title does not match the results. Although the work demonstrates that the WFA lectin is capable of binding to proteins expressed in cancer cells resistant to TAM, it is recommended to carry out a statistical correlation analysis between positivity with WFA and the presence of resistance to TAM, and/or the clinical- pathological characteristics of the patients. One option is to change the job title, the suggestion would be:

TAM-resistant breast cancer cells react positively to the lectin WFA.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Veronica Ivonne Hernández Ramírez

**********

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5 Aug 2022

July 07, 2022

Dear Editor,

We very much appreciate the Reviewers’ and your careful reading and evaluation of our manuscript, as well as your invitation to resubmit our work after appropriate revision. Based on all your comments, we have revised our manuscript and are re-submitting it herewith. Our point-by-point responses to all your comments are provided below, highlighted in Arial font. All revisions in the text are in red font.

Response to Editors and Reviewers:

Reviewer #1: The topic on glycans for drug resistant in breast cancer treatment is interesting. However, the data current cannot support the finding and conclusions.

1. Glycomics has been studied in other reports, where the modification sites and glycan structures

can be clearly identified. The authors carried out mass spectrometry analysis but did not give out any

meaningful proteomic data (probably only identify some protein sequences).

>As the Reviewer mentioned, for example, a study that we cited as reference 16 (J Proteomics 2021) revealed data on changes in glycan structure observed in paclitaxel-resistant cells. However, to our knowledge, hormone therapy resistance has not been investigated in detail. Nevertheless, the purpose of our study was not to reveal meaningful proteomic data, but to identify differences in glycan expression in TAM-resistant cells. We apologize for any misleading expressions in the text. We have corrected some descriptions in the Abstract and Discussion section to avoid confusion among readers. We hope these comments and revisions will satisfy the Editors and Reviewers.

2. Lectin array can only give the subtype of glycoprotein expression and it should be integrated with

protein expression (WB) to see the glycosylation change.

>Our data could mean either a change in glycosylation or a change in expression of the glycoproteins themselves. To further investigate these points, expression and binding by immunoprecipitation with the lectins and candidate proteins should be tested. We tried several commercially available antibodies for immunoprecipitations and western blot but unfortunately they did not work well in these experiments. Therefore, the Reviewer's point is yet to be answered, but we still believe that our current data will be of interest to your readers.

3. Clinical sample analysis is not very related cell-based study, which need more data to support (e.g. quantitative glycoproteomics on the clinical samples).

>As mentioned above (question 1), we could not manage to identify specific glycoprotein(s). Therefore, further clinical sample analysis, which was suggested by the Reviewers, could not be performed. Glycoproteomic analysis of clinical samples is a subject of our future work and will hopefully be published as a separate paper.

Reviewer #2:

Line 94: resistant cell lines are described as if they had been developed in the laboratory itself and this is not the case, they were acquired from a commercial company.

>We apologize for our confusing explanation. The resistant cells were established in our laboratory. We have corrected the description in the Methods section.

Line 100: the term "parental cells" does not apply, although it is the same cell line with acquired resistance, they do not come from the same cell passage

>We have now revised our manuscript throughout. “Parental/parent” has been replaced with “sensitive” although there may be some confusion by the reviewer as stated above.

Line 256: the proteins identified in the WFA-binding fraction in TAM-cells are mentioned, nevertheless the same analysis should be carried out in the cells not TAM to be able to conclude with greater support the results

>We appreciate the reviewer’s suggestion. Binding to WFA was elevated only in TAM-resistant cells, and it was not possible to extract WFA-binding glycoproteins by affinity-isolation with WFA from TAM-sensitive cells. We realized that there were some common proteins detected in the two TAM-resistant cell lines. Therefore, it was not possible to carry out “the same analysis” with TAM-sensitive cells.

Line 264: It is not clear whether the control group refers to patients treated with tamoxifen who did not develop metastases or rather who did not relapse.

>We meant those who did not develop metastases. We have now revised the sentence to “Primary tumors from patients who developed distant metastasis during adjuvant TAM treatment (n = 20) and those who did not develop recurrent disease (n = 20) were analyzed”.

Line 307: is incorrectly worded, lectin binding to glycoproteins does not contribute to resistance, but rather could function as a potential biomarker or prognostic factor.

>We appreciate the Reviewer pointing this out. We have now revised the statement to “When comparing WFA staining intensity in metastatic lesions that developed during TAM treatment, there was no difference in the expression level compared with their paired primary lesion…”.

Reviewer #3: In the methods section, the identification of lectin-bound proteins should be mentioned in a single section.

>We appreciate the Reviewer’s suggestion. We have now revised the Methods section under a new title “Preparation of WFA-binding proteins and identification by liquid chromatography/mass spectrometry (LC/MS) followed by database search using Mascot”.

In the methods section, the quantification of the biotinylated lectin reaction in tissue from cancer patients is deficient. It is suggested to explain in detail. Consider published methods to assess different levels of positivity.

1) Diagn Pathol. 2014 Nov 29;9:221.doi: 10.1186/s13000-014-0221-9.Different approaches for interpretation and reporting of immunohistochemistry analysis results in the bone tissue - a review. Nickolay Fedchenko 1 2, Janin Reifenrath 3. DOI: 10.1186/s13000-014-0221-9

2) Pathol Res Pract. 2015 Dec;211(12):973-81.doi: 10.1016/j.prp.2015.10.002. Epub 2015 Nov 6. Integrins and haptoglobin: Molecules overexpressed in ovarian cancer. Julio César Villegas-Pineda 1, Olga Lilia Garibay-Cerdenares 2, Verónica Ivonne Hernández-Ramírez 3, Dolores Gallardo-Rincón 4, David Cantú de León 5, María Delia Pérez-Montiel-Gómez 6, Patricia Talamás-Rohana 7

3) Cancer Cell Int. 2022; 22: 6. doi: 10.1186/s12935-021-02425-6 PHD finger protein 20-like protein 1 (PHF20L1) in ovarian cancer: from its overexpression in tissue to its upregulation by the ascites microenvironment. Dulce Rosario Alberto-Aguilar,1 Verónica Ivonne Hernández-Ramírez,1 Juan Carlos Osorio-Trujillo,1 Dolores Gallardo-Rincón,2 Alfredo Toledo-Leyva,2 and Patricia Talamás-Rohana1

>We appreciate the reviewer’s suggestion. First, we simply semi-quantitatively assessed the WFA expression in surgical specimens without relying on any scoring system. We have now added the necessary description in the Methods section and legends for Figure 4.

We thank the reviewer for giving us useful information from some previous studies. Unfortunately we could not manage to conduct such a detailed assessment as that performed by Dulce Rosario Alberto-Aguilar et al (Cancer Cell Int. 2022), but now we are well aware of the need to perform further studies to obtain more quantitative results. We have now added this issue as a limitation of the current study in the Discussion section (lines 315-317). We hope these comments and revisions will satisfy the Editors and Reviewers.

The title does not match the results. Although the work demonstrates that the WFA lectin is capable of binding to proteins expressed in cancer cells resistant to TAM, it is recommended to carry out a statistical correlation analysis between positivity with WFA and the presence of resistance to TAM, and/or the clinical- pathological characteristics of the patients. One option is to change the job title, the suggestion would be: TAM-resistant breast cancer cells react positively to the lectin WFA.

>We appreciate the reviewer’s suggestion. We have now changed the title as suggested. Because the term WFA (Wisteria floribunda agglutinin) includes “lectin”, we decided to use the revised title “TAM-resistant breast cancer cells exhibit reactivity with Wisteria floribunda agglutinin”.

Additional Editor Comments:

I have recommended major revision for your manuscript, based on the comments and observations made by three reviewers. Although your manuscript presents primary research that contributes to scientific knowledge, it requires additional work because publication criteria 3 and 4 were not completely covered.

Criteria No. 3. Experiments, statistics, and other analyses are performed to a high technical standard and are described in sufficient detail.

The three reviewers are asking for further methodological descriptions. Moreover, I consider that the inclusion of the MS analysis of sensitive cells should be included as control.

In addition, bioinformatic analysis can be performed in order to determine whether the identified proteins contain the N-acetylgalactosamine beta 1 (GalNAc beta 1-3 Gal) modification that is recognized by the WFA lectin.

>Methodological descriptions have been revised accordingly.

We judged it was not possible to conduct MS analysis of the sensitive cells as a control, due to the very low binding level of WFA in the sensitive cells, as shown in Figure 2.

We appreciate your suggestion of further bioinformatic analysis. However, we were not able to identify the specific glycoproteins that bind to WFA in this study. Therefore, we could not perform the analysis you suggested. We investigated the relationship between the expression of B3GALNT2, a GalNAcbeta1-3GlcNAc glycosyltransferase which is responsible for the biosynthesis of the LacdiNAc structure having high affinity with WFA (reference 28), and the prognosis of patients who were treated with TAM, employing a data base available in the public domain (Kaplan-Meier plotter). The results showed that patients with B3GALNT2-high tumors had significantly worse outcomes. The results of this analysis have been added as Supplementary Figure 5 and have been mentioned in the revised Discussion. Although these indirect observations should be interpreted with caution, we believe that the specific glycan structures recognized by WFA merit further investigation in relation to resistance to TAM. We hope these comments and revisions will satisfy the Editors.

Criteria No. 4. Conclusions are presented in an appropriate fashion and are supported by the data.

The manuscript does not contain any experimental results that confirm the role of the identified glycoproteins in the TMX resistance mechanism. For this reason, reviewer No. 1 rejected the manuscript and reviewer No. 3 is proposing a change in the title of the manuscript. Therefore, authors have to decide if they go deeper in looking for the participation of the glycoproteins identified in the resistance mechanisms or discard this asseveration from the title.

>We appreciate the Editor’s suggestion. Because we were unable to determine the specific glycoproteins, we have changed the title as suggested.

We again thank you for your consideration. We look forward to hearing from you at your earliest convenience.

Yours sincerely,

Yoshiya Horimoto

10 Aug 2022

Tamoxifen-resistant breast cancer cells exhibit reactivity with Wisteria floribunda agglutinin.

PONE-D-22-13506R1

Dear Dr. Horimoto,

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Additional Editor Comments (optional):

Reviewers' comments:

16 Aug 2022

PONE-D-22-13506R1

Tamoxifen-resistant breast cancer cells exhibit reactivity with Wisteria floribunda agglutinin.

Dear Dr. Horimoto:

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