Background
Breast cancer (BCA) is one of the global public health problems. According to the International Agency for Research in Cancer Fact Sheet, 2018 showed that BCA in approximately 2,088,849 people year, was in the top rank of new cancer cases worldwide [
1]. Regularly, the mortality of cancer is attributed to various processes in cancer progression, including metastasis, proliferation and chemoresistance. These processes were influenced by not only the properties of cancer cells themselves but also to the effects of the tumor microenvironment (TME). TME is therefore now considered a therapeutic target [
2]. Periostin (PN) is one of active molecules in TME that has been reported to be a promoter of cancer progression through various mechanisms including proliferation, invasion/migration, angiogenesis and chemoresistance [
3,
4]. PN expression is up-regulated in the TME of many types of cancer, for example, non-small cell lung cancer (NSCLC) [
5], malignant pleural mesothelioma [
6], prostate cancer [
7], renal cell carcinoma [
8], cholangiocarcinoma (CCA) [
9] and also BCA [
10]. Regularly, PN molecules are secreted from cancer-associated fibroblasts (CAF) and can activate the cancer cells via integrin receptors [
3]. PN expression in cancer tissue was significantly correlated to survival of patients in colorectal cancer (CRC) [
11], CCA [
9] and BCA [
12]. In addition, serum PN was also positively correlated with poor prognosis in CRC [
13] and CCA [
14]. In BCA, however, the correlation between patient survival and serum PN is still controversial since no correlation [
15] or strong correlation [
16] have been reported. PN is also related to chemoresistance of cancer cells, usually via PI3K/Akt/survivin signaling to increase cell viability in the presence of chemotherapeutic agents [
17,
18]. Chemoresistance is one of the main difficulties for cancer treatment that can lead to mortality of the patients, therefore, the understanding and manipulation of chemoresistance can support patient survival.
PN has been reported to be associated with many kinds of chemotherapeutic drugs, such as arsenic trioxide in hepatocellular carcinoma (HCC) [
19]; methotrexate, doxorubicin [
20], cisplatin [
21], carboplatin and paclitaxel [
22] in ovarian cancer (OVC); oxaliplatin and 5-fluorouracil (5-FU) [
18] in CRC and gemcitabine in pancreatic cancer [
23]. Moreover, PN was also shown to be associated with anti-angiogenic therapy [
24]. It was found that inhibition of PN can restore the sensitivity to chemotherapeutic drugs [
25]. Since, almost cytotoxic drugs have serious adverse effects that can lead to morbidity and mortality [
26], a minimum of drug usage should be of benefit to cancer patients. To overcome chemoresistance from PN, a bioactive peptide is one of the interesting molecules to develop an inhibitory mechanism [
27]. A bioactive peptide can be defined as a peptide that can bind to a molecular target and has an effect on cells or organisms. It has advantageous properties for use in cancer therapy such as high tissue penetration as compared to full size antibodies, good biocompatibility and binding affinity to target molecules [
27]. There are many peptides used for therapeutic purposes including cancer treatment [
28]. To date, the database web site
http://crdd.osdd.net/raghava/thpdb/ [
29] reported 61 peptides approved by United States Food and Drug Administration (US-FDA) for cancer therapy [
30]. Peptides can be used as a high-throughput screen for many methods including phage biopanning from the peptide library [
27].
In this study, a 12 amino acid peptide library was used for screening of bioactive peptides that could bind to PN at an integrin binding site [
31] and inhibit PN function of BCA cells in in vitro experiments. In addition, screening of PN expression in cancer tissue and measurement if serum PN levels from BCA patients were performed and the correlation analyzed with clinical elements including the response to anthracycline-based chemotherapy. The application of this peptide may be used in future clinical practice to restore the sensitivity to chemotherapeutic drugs in BCA cells and reduce the dosages in patients which could decrease the morbidity and mortality from the adverse effects of the drug.
Methods
Cell cultures of BCA cells
BCA cell lines, MDA-MB-231 and MCF-7 were used in the study. They were cultured with Dulbecco Modified Eagle’s Medium (DMEM) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). These media contained 10% fetal bovine serum (FBS) (Gibco), using penicillin/streptomycin (Gibco) as antibiotics and amphotericin B (Gibco) as an antifungal drug with 5% CO2 and 90% humidity at 37 °C.
Lipofectamine™ 3000 (Invitrogen, Thermo Fisher Scientific) was used to transfect the blank pCDNA™3.1 plasmid (v385–20, Invitrogen) or pCDNA™3.1 PN-plasmid into BCA cell lines. After transfection, the cells were selected by Geneticin™ (Gibco) (up to 1 mg/ml) to create stable cell lines. PN and integrin expressions were tested by reverse transcriptase (RT)-polymerase chain reaction (PCR) using Light Cycler® 480 II system (Roche, Basel, Switzerland) with specific primers (Table
1) [
9,
32]. Glyceraldehyde-3-phosphate dehydrogenase (
GAPDH) mRNA expression was used as an internal control (Table S
1). The cycle threshold (Ct) value was used for calculation of expression folding.
Western blot analysis
To determine the secreted PN amount in conditioned-media of each cell, 3 × 105 cells were seeded into 6-well plates with 3 ml of complete media. Cell media were removed the next day and washed with phosphate-buffered saline (PBS), and serum free media was added. Media was collected at 24 h, centrifuged and supernatant was taken and concentrated by Vivaspin®6 (VS0691, Sartorius, Goettingen, Germany). Protein concentration was determined by Bradford reagent (#5000006, Bio-Rad Laboratories, Hercules, CA, USA) and the amounts were adjusted to 5 μg per loading. To determine protein expression or phosphorylation in the cellular part, 2 × 106 cells were lysed with RIPA buffer (sc-24,948, Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Each sample was separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and transferred to PVDF membrane. The membranes were blocked with 5% bovine serum albumin (BSA) and immunodetection for PN was continued with goat anti-PN polyclonal antibody (sc49480, Santa Cruz Biotechnology) and rabbit anti-goat IgG conjugated with horseradish peroxidase (HRP) (HAF017, R&D Systems, Minneapolis, MN, USA) for PN detection, rabbit anti-Akt polyclonal antibody (#9272, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-pAkt polyclonal antibody (#9271, Cell Signaling Technology), rabbit anti-survivin polyclonal antibody (#2803, Cell Signaling Technology) and goat anti-rabbit-HRP (ab6721, Abcam, Cambridge, UK) for AKT, phosphorylated AKT (pAKT) and survivin. β-actin expression was determined as an internal control for the cellular part using mouse anti-β-actin polyclonal antibody (sc47778, Santa Cruz Biotechnology) and horse anti-mouse-HRP (#7076, Cell Signaling Technology). HRP was detected by Pierce™ enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific) and chemiluminescence signal was detected by G:BOX gel documentation system (Syngene, Cambridge, UK). Expression level of PN was determined by ImageJ version 1.52a software (National Institutes of Health, Bethesda, MD, USA).
Immunocytochemistry
BCA cells, approximately 1 × 105 cells, were plated on top of coverslips in 24-well plates and cultured for 24 h. After that, media was removed and cells were washed with PBS, fixed with 4% paraformaldehyde, washed again and blocked with 5% FBS. The primary antibody used was mouse anti-human integrin αVβ5 monoclonal antibody (MAB2019Z, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), at a concentration of 15 μg/ml and incubated with the cells at 37 °C for 4 h. After that, cells were washed with PBS and incubated with Cy™3 AffiniPure F (ab’)2 Fragment Goat Anti-Mouse IgG, Fcγ fragment specific (115–166-071, Jackson Immuno Research Inc., West Grove, PA, USA), at a concentration of 0.5 μg/ml, at room temperature (RT) for 1 h. Hoechst 33258 was used for nuclear staining. Confocal imaging experiments were conducted on a Zeiss LSM 800 (Carl Zeiss, Jena, Germany) at the Division of Molecular Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University. Equipment details were microscope model: AxioObserver7, objective lens: Plan-Apochromat 63x/1.4NA oil immersion and laser: Diode 561 nm. Acquisition software was ZEN 2.3 software (blue edition, 2002–2011).
Serum and tissue specimens
Serum was obtained from left-over specimens of pre-operative BCA patients and normal females who came for health checks for determination of PN concentrations. Cancer tissues from BCA patients were taken from the remaining tissue after pathological examination. Serum and tissues and clinical information collections were performed by Prof. Pornchai O-charoenrat, Department of Surgery, Faculty of Medicine Siriraj Hospital, Mahidol University, under agreement of Siriraj Institutional Review Board No. Si519/2010.
Phage biopanning
Ph.D.™-12 phage display peptide library (New England Biolabs, Ipswich, MA, USA) was used for phage biopanning of anti-PN peptide following the manufacturer’s protocol. In summary, a peptide fragment correlated to the integrin binding site of PN [
31] conjugated with biotin (Biotin-ERIMGDKVASEALMKYHILN) was added into Pierce™ Streptavidin Coated High Capacity Plates (#15500, Thermo Fisher Scientific). Continuously, 10
10 plaque forming units (pfu) of bacteriophage were poured into the wells and incubated at RT for 1 h. Unbound phages were then washed off and bound phages were collected. Phage titering was continued using
E. coli strain ER2738 (New England Biolabs). The bacteria were centrifuged, and supernatant with virus was kept in a fresh tube. Phage precipitation was performed by adding of 1/6 volume of NaCl/polyethylene glycol solution (20% w/v PEG-8000 with 2.5 M NaCl). After that, phage titering was observed on LB/IPTG/Xgal plates, and the amplified phages were used for next round. In this way, the panning process was repeated seven times. During the biopanning process, a negative selection for phage clones was also performed to exclude streptavidin and plastic binding phage. Twenty phage clones per round from third, fifth and seventh rounds were randomly selected for DNA sequencing. Selection of candidate phage clones was done according to the results of sequencing. The sequence with highest frequency was assumed as the best phage clone to be used for further experiments. The sequences were also checked by online database to target unrelated peptides (
http://i.uestc.edu.cn/sarotup3/index.html) [
33] and to identify and rule out the peptide sequences which had high probability of binding to streptavidin and plastic more than 0.5. The binding affinity of selected phage clones were confirmed by the dot blot method. Volumes of 1 μl with 500 ng of recombinant PN (rPN) (RD172045025, BioVendor, Brno, Czech Republic) or BSA were spotted on nitrocellulose membranes, dried for 15 min and placed in 96-well plates then blocked with 5% BSA. Membranes were incubated with the selected phage or blank phage clones (10
12 pfu in 50 μl) at 4 °C overnight. Then membranes were washed and incubated with 50 μl (2 μg/ml) of anti-M13 antibody-HRP (ab50370, Abcam) at RT for 1 h and detected by ECL.
Peptide design and synthesis
After selecting the best binding sequence of 12-amino acids peptides, 2 types of peptide would be synthesized, plain peptide and peptide conjugated with fluorescein isothiocyanate (FITC). For the synthesis of the latter, a spacer region (GGGSCK) would be added at the C-terminal end of the peptide and FITC was conjugated with the side chain of lysine. Finally, C-terminal amidation would be performed. The synthesis of plain and FITC-labelled anti PN peptides was ordered from Syn Peptide company (Shanghai, China). FITC-labelled anti-PN peptide tested binding affinity to non-denaturing cell lysate of transfected BCA cells and their mock transfected cells and rPN by dot blot analysis. Briefly, 12.5 μg of cell lysate or 500 ng of rPN in 1 μl was applied onto nitrocellulose membrane. The membrane was blocked with 5% BSA followed by peptide incubation at 4 °C overnight and the fluorescent signal was detected the next day using the G:BOX gel documentation system. The checking of anti-PN peptide binding to intact PN-transfected BCA cells was also performed with similar process as immunocytochemistry plus a step of cell membrane permeabilizing after fixation by incubated with 1% Triton X for 1 min at RT. The single staining step was done by incubation of the permeabilized cells with 2 μM FITC-labelled anti-PN peptide for 1 h at RT, washed and then nuclear stained with Hoechst 33258. The observation was viewed under confocal microscope using laser diode 488 nm.
Determination of peptide binding affinity
The physical properties of this peptide would be determined by an online tool (
https://www.thermofisher.com) (Thermo Fisher Scientific). Structure of this peptide and the binding properties to PN would be predicted by PEP-FOLD3 [
34] and pepATTRACT [
35] tools from RPBS Web Portal (
https://bioserv.rpbs.univ-paris-diderot.fr) using PN 3D-structure from RCSB PDB database (
https://www.rcsb.org/structure/5YJG) [
36]. Binding affinity of anti-PN peptide was determined with isothermal titration calorimetry [
37] using the MicroCal PEAQ-ITC Machine (Malvern Panalytical Ltd., Malvern, UK) in which 700 nM of rPN (or BSA as negative control) and either 70 nM of commercial goat anti-PN polyclonal antibody or 100 nM of plain anti-PN peptide in 50 mM Tris and 150 mM NaCl (pH 7.5) buffer were added into syringe and cell compartments of the machine. The procedure followed the instructions for the machine. The results would be determined by the measurement of the exothermic energy after intermittent injection into the cell compartment and reported as the binding affinity constant (KD).
Proliferation assay
The proliferation assay of BCA cells was determined by the cell viability assay using CellTiter 96® Aqueous One Solution Cell Proliferation Assay (MTS assay) (Promega, Madison, WI, USA). Briefly, 3 × 103 cells in 100 μl medium were seeded in 96-well plates for 24 h. The day 1 baseline determination used the MTS assay following company instructions. For the experiment, cells were placed into new media containing 2% FBS with or without rPN (100 ng/ml) and anti-PN peptide (1 μM) and cells were cultured for a further 72 h. After treatment, cell viability was measured by the MTS assay. Proliferation rate was calculated as the folding of cell numbers increased from the baseline.
Determination of cell stemness in PN-transfected cell and effect of anti-PN peptide
Stemness of PN-transfected BCA cells was determined by staining with anti-CD24 and anti-CD44 antibodies and analyzed by flow cytometry and compared with mock-transfected cells in conditions without or with anti-PN peptide. Briefly, BCA cells were seeded at the concentration 1 × 105 cells into 6-well plates with 2 ml of complete media. Cell media was removed the next day and changed to 1% FBS media for 24 h. Then, the treatment in 1% FBS media without or with anti-PN peptide (1 μM) were refilled and incubated for a further 24 h. At the end of treatment, cell pellets were collected and incubated in 2% FBS/1x PBS with 1:20 dilution of FITC-labeled anti-CD24 antibody (cat no. 21270043, ImmunoTools GmbH, Friesoythe, Germany) and 1:5 dilution of allophycocyanin (APC)-labeled anti-CD44 antibody (cat no. 21270446, ImmunoTools) for 30 min. CytoFLEX® Flow cytometry (Beckman Coulter, Inc. Brea, CA, USA) and CytExpert® software version 2.1 (Beckman Coulter, Inc.) were used for analysis.
Determination of the half-maximal inhibitory concentration (IC50)
Determination the IC50 of BCA cells in response to chemotherapeutic drugs was analyzed by MTS assay. The experiment started from 5 × 103 of BCA cells in 100 μl medium that were seeded in 96-well plates for 24 h, then media was changed with different concentrations of new chemotherapeutic drugs [doxorubicin (S1208), paclitaxel (S1150), cisplatin (S1166), 5-fluorouracil (5-FU) (S1209) and gemcitabine (S1714) that were purchased from Selleck Chemicals, Houston, TX, USA] with or without rPN and anti-PN peptide for a further 48 h. After that, cell viability was determined by the MTS assay. Combination index (CI) was calculated by IC50 of combination of drug and PN or anti-PN peptide divided by IC50 of chemotherapeutic drug alone.
Migration assay
The wound healing assay was performed to determine migration activity of the BCA cells after treatment under various conditions. Briefly, 5 × 10
4 cells of parental BCA cells or mock/PN-transfected cells were seeded in 24-well culture plates with their regular media for 24 h and media changed to 1% FBS with or without rPN (100 ng/ml) and anti-PN peptide (1 μM) and culture continued for 1 day until they reached approximately 95% confluency. Wounds were applied by scratching using 200 μl micropipette tip in a single straight line. The media with floating cells would be removed and refilled with the new experimental media. The culture process would be continued for a further 24 h and photos of the area would be taken at 8 h intervals. The migration area would be analyzed from the photos taken using TScratch version 1.0 software (
https://github.com/cselab/TScratch) [
38] and migration activity calculated as μm
2/h. Independent duplicated experiments were performed.
Enzyme-linked immunosorbent assay (ELISA) for serum PN measurement
Periostin ELISA Kit (Human) (Shino-Test Corporation, Tokyo, Japan) was used for measurement of serum PN following the procedure as previously described [
14].
Immunohistochemistry
Matching tissues with the serum PN measurement from BCA patients were used for PN staining. An immuno-peroxidase staining procedure was performed using the method in paraffin-embedded BCA tissues as previously described. The area and intensity of expression was estimated and semi-quantitatively graded as 0–3 scores. For area determination, 0 was up to 5%, 1 was 6–25%, 2 was 26–50% and 3 was 51–100% of either the cancer or fibroblast area. For intensity scoring, 0 was negative, 1 was weakly positive, 2 was moderately positive and 3 was strongly positive when compared to positive and negative controls. Quick score (Q-score) was determined by multiplying the area and intensity scores. For statistical analysis, the scores of 0–4 were categorized as low expression, and 6–9 as high expression.
Statistical analysis
ANOVA test or Student’s
t-test were applied for statistical analysis of the experiments. GraphPad Prism® version 7.04 software (GraphPad Software, San Diego, CA, USA) was used in calculation of IC50 values of chemotherapeutic drugs and PASW Statistics software version 18 (SPSS, IBM, Armonk, NY, USA) was used for other analysis. Dose-responses were compared by Holm-Sidak’s multiple comparison test. Mann-Whitney ranked-sum test was used for comparisons between normal and patient serum PN. Chi-square or Fisher’s exact test were used to determine the correlation between PN expression and clinical data in BCA patients. Kaplan-Meier Log-rank test using Kaplan-Meier Plotter online tool (
https://kmplot.com/) was performed to determine the correlation between PN expression and survival time of BCA patients by online database [
39].
P-values of < 0.05 were used as statistical significance.
Discussion
The latest Global Health Observatory data from the World Health Organization in 2016 showed that BCA is the 8th cause of death of females worldwide [
42]. Even though there are many ways for early detection of BCA, many patients need chemotherapy. Doxorubicin or adriamycin is one of the first line chemotherapeutic agents in BCA patients [
43]. It is a member of the anthracycline family of chemotherapeutic agents. It can intercalate double stranded DNA and inhibit topoisomerase II enzyme activity that then suppresses DNA replication and causes cytotoxicity [
43]. Similar to almost cytotoxic drugs, doxorubicin also has serious adverse effects, especially immunosuppression and cardiotoxicity that can cause mortality in patients [
44]. To overcome these adverse effects, nanoparticle formulation was introduced in the clinical use of doxorubicin [
43], however, the cost of these formulas was much increased too.
This study successfully generated PN expression BCA cell lines and checked the expression of the PN receptor, integrins. All cells presented integrins expression especially αVβ5 heterodimer, which can act as PN receptor [
3,
31], similar to the previous article for both commercial cell lines, MDA-MB-231 and MCF-7 [
45]. This proved that these cells can respond to PN in an autocrine-paracrine manner. PN-transfected cells were used to screen for drug sensitivity. These results determined that only doxorubicin resistance was present in the current system. Doxorubicin has been reported in resistance in MDA-MB-231 and MCF-7 cell lines by in vitro experiments that showed resistance was mediated in the extracellular matrix [
46]. Moreover, higher PN expression was presented in induced doxorubicin resistant W1 ovarian cancer cell lines compared with the parental cells [
20]. This evidence can confirm present results that PN can induce doxorubicin in cancer cells. In addition, PN showed promotion of proliferation and migration in all BCA cells (Fig.
2), that corresponded to previous studies reported in CCA cells [
9]. To reverse these effects, anti-PN peptide was designed to counteract PN on cancer promotion activities.
Only one anti-PN peptide against the integrin binding site was screened from the bacteriophage library. It showed affinity binding to PN, similar to commercial anti-PN antibody (Fig.
1). Physical properties of this peptide show stability in extracellular environment which is the location of secretory PN (Fig. S
4). Peptide structure was showed as linear with small alpha helix (Fig. S
5a) and bind to PN at active site (Fig. S
5b). It could also inhibit PN-induced cell proliferation and migration in BCA cells, with minimal effect on cells without PN (Fig.
2). This evidence confirmed that the inhibitory effects were from binding to PN but not directly to the cells. The inhibition of PN-induced proliferation was shown better in rPN treatment than in the PN-transfected experiment; this may be that prolonged exposure of PN had more effect than short time treatment in the proliferation assay. For reversing of the doxorubicin resistance induced by PN, anti-PN peptide shifted the dose response curve to the left in PN presenting conditions (Fig.
3). This result indicated the effect of anti-PN peptide to improve drug resistance in BCA with PN expression. The results of the present study also demonstrated that the intracellular signaling mechanism might be via the phosphorylation of Akt and downstream expression of survivin (Fig.
4) as in previous reports [
17,
18], therefore, this may also be considered as a target of repression of PN action in cancer promotion. Stemness has been recognized as a molecular mechanism of chemoresistance including in breast cancer [
47]. This report also determined survivin as an upstream molecule of stemness. In addition, PN has been also recognized as a cancer stemness marker too [
48]. Our results showed higher CD24
−/CD44
+ stem cell in PN-transfected MCF-7 compared with mock control and it could be reduced by anti-PN peptide. MDA-MB-231 BCA cell has also shown the similar pattern but less different than MCF-7 according to the baseline of stemness in MDA-MB-231 was much higher than MCF-7. Unfortunately, the statistical test was not significant according to minimally change in MDA-MB-231 cell and high error bar in MCF-7 cell. A further study might be in an in vivo system toward implementation for clinical usage. Since anti-PN antibody was studied to inhibit PN-promoted cancer progression in mouse model [
49], anti-PN peptide should have an advantage providing more ability for tissue penetration [
50].
In proliferation experiments, anti-PN peptide showed inhibition only in PN treatment but seemed not in PN-transfected experiment. The explanation might start that PN actions as paracrine in almost studies [
3,
4]. However, some reports showed that some cells such as keratinocytes, renal mesangial, renal tubular epithelial cells and BCA cells expressed PN but not secreted [
51]. Moreover, few studies indicated the localization of PN in cytoplasm and nucleus of BCA (MCF-7 and MDA-MB-468) and COS7 cells [
52,
53]. In addition, some cytokines showed the function in both extracellular and intracellular, such as IL33 and HMGB1, for examples [
54,
55]. A report showed that overexpression renal mesangial cell had increasing of proliferation and fibronectin secretion without evidence of extracellular PN [
51]. The other study indicated the knockdown of either integrin αVβ3 or PN in lung cancer cell could reduce cell proliferation and double knockdown showed much lower [
56]. These phenomena might be the explanation of intracellular PN function on cell proliferation that anti-PN peptide could not or minimally inhibit the cell proliferation in BCA cells. BCA cells including MCF-7 and MDA-MB-231 had been reported the minimal expression of PN [
57]. In this study, the overexpressed PN could be secreted and detected in all PN-transfected BCA cells. In addition, the intracellular signaling via pAKT and survivin could be inhibited by anti-PN peptide. However, if the transfection of PN plasmid which was not the natural condition could introduce some intracellular function of PN, therefore, anti-PN peptide could not inhibit this mechanism. This phenomenon was showed only in proliferation but not migration assay which might have different regulation. To answer this question, the further experiments such as manipulation of PN secretion by inhibitor and examine the proliferation/migration activity should be performed.
Eighty-three BCA patients with tissue and serum specimens were included in this study. Immunohistochemistry in this study’s setting did not show staining of PN in cancer cells, neither in the previous studies in CCA [
9,
14], while signals in the stroma area were very strong (Fig.
5). In addition, the results from real time RT-PCR of both BCA cell lines showed high baselines of Ct values (approximately 32 and 39 for MDA-MB-231 and MCF-7, data not shown), implying that the expression was low. For MCF-7, the Ct value was equal to a previous report [
52]. Immunohistochemical results, however, did not correlate with the previous reports [
52,
57]. The variation might be from the antibody, staining conditions, background and also different sample groups. The results were confirmed for only stromal staining without being positive in cancer cells. The results showed that tissue PN expression and serum PN level had a significant correlation, however, only 36.2% of cases had strong positive of PN staining and only 10.8% had a high level of serum PN. The median of serum PN levels in BCA patients did not differ from normal controls, but the maximum was higher. Analysis by online database showed significant correlation between PN protein expression and poor survival of BCA patients (Fig.
6d, e), similar to recent publication [
16]. However, in this study, there was no correlation between serum PN and clinical data. This finding supported a previous study in early BCA [
15]. While the other report showed opposite [
57]. The explanation might be that the expression of PN in that publication was in cancer cells but the present result was in stroma cells, so that the results could be different. Since prognosis of treatment in this group of BCA patients was good, there were only 32 patients (38.6%) received an anthracycline (doxorubicin or epirubicin), so it may be a lot of parameters that could interfere the anthracycline-based treatment. Taken together, this indicated that PN seemed not to be significant in the all-over BCA picture but might be important in some situations. Unlike CCA [
9,
14], PN have no clinical significance in BCA may because of the small size of the cancer tissue, compared with CCA.
Conclusions
This study established a new anti-PN peptide that could counteract PN-mediated cancer progression including induced doxorubicin resistance. This peptide could be developed for clinical usage in the future, not only for BCA, but also for other cancers such as CCA.
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