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Wnt/β-catenin pathway is a key signaling pathway to trastuzumab resistance in gastric cancer cells

Wnt/β-catenin pathway is a key signaling pathway to trastuzumab resistance in gastric cancer cells Background Trastuzumab is the only approved target agent for the first-line treatment of human epidermal growth factor receptor-2 (HER-2) positive gastric cancer; however, trastuzumab resistance is a major problem in clinical practice. To comprehend the mechanism of trastuzumab resistance, we focused on the Wnt/β-catenin signaling pathway and its influence on the phenotypes and behavior of trastuzumab-resistant gastric cancer cells. Methods Trastuzumab-resistant NCI-N87R cells were established in vitro from the human gastric cancer cell line NCI-N87 by dose-escalating repeated trastuzumab treatment. We investigated the phenotypes of NCI-N87R cells, including Wnt signaling pathway activity. Gastric cancer organoid cells were incubated with complete medium and Wnt3a-depletion medium, and their resistance to trastuzumab was compared. Results NCI-N87R exhibited stemness and epithelial-mesenchymal transition (EMT )-like phenotypes, along with decreased levels of the epithelial marker E-cadherin and increased levels of the mesenchymal markers Vimentin and Snail along with an increased Wnt signaling pathway activity. When gastric cancer cells were incubated in Wnt3a- conditioned medium. Wnt signaling pathway activity and resistance to trastuzumab increased. Gastric cancer patient- derived organoids incubated in Wnt3a-depletion medium were more susceptible to dose-dependent inhibition of cell viability by trastuzumab than those incubated in complete medium. Conclusions Trastuzumab-resistant gastric cancer cells exhibited EMT-like phenotype, and trastuzumab resistance was promoted by the Wnt/β-catenin signaling pathway. The Wnt/β-catenin pathway is a key signaling pathway for trastuzumab resistance in gastric cancer cells. Keywords Wnt, Gastric cancer, Trastuzumab, Resistance, Epithelial to mesenchymal transition † Department of Pathology, Yonsei University College of Medicine, Yuna Kim and Yoo Jin Bae These two authors contributed equally Seoul 03722, Korea to this work. Department of Pathology, Gangnam Severance Hospital, Yonsei *Correspondence: University College of Medicine, Seoul 06229, Korea Jie-Hyun Kim Department of Internal Medicine, Division of Gastroenterology, Yonsei otilia94@yuhs.ac University College of Medicine, Seoul 03722, Korea Department of Internal Medicine, Division of Gastroenterology, Gangnam Severance Hospital, Yonsei University College of Medicine, 20, Eonju-ro 63-gil, Gangnam-gu, Seoul 06229, Korea © The Author(s) 2023. 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The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Kim et al. BMC Cancer (2023) 23:922 Page 2 of 8 Introduction Materials and methods Gastric cancer ranks fifth in incidence and fourth in Gastric cancer cell line and culture mortality globally, with an estimate of one million new Gastric cancer cell lines including MKN45, MKN74, cases and 769,000 deaths reported in 2020 [1]. Human SNU216, SNU484, NCI-N87, and AGS were obtained epidermal growth factor receptor-2 (HER-2), a member from and authenticated by the Korean Cell Line Bank of the epidermal growth factor receptor (EGFR) family, with STR profiling. All cells were cultured in the RPMI is an important treatment target for gastric cancer [2]. 1640 medium that had 10% FBS added to it at 37 °C and Amplification of the HER2 gene is observed in approxi - 5% CO in a humid incubator. mately 20% of patients with gastric cancer [3, 4]. Previ- ous studies have suggested that HER-2 overexpression Preparation of conditioned medium is positively associated with cancer cell proliferation, Wnt3a-conditioned medium was harvested from Wnt3a malignancy, metastasis, and unfavorable outcomes [2, expressing L-cells (CRL-2647; American Type Culture 5, 6]. HER-2 overexpression has also been observed in Collection, Manassas, VA) according to the manufac- other solid tumors, including biliary tract, colorectal, turer’s instructions. Wnt3a-conditioned medium was non-small-cell lung, and bladder cancers [7–10]. Treat- depleted of the Wnt3a protein by incubation with 4  µg/ ment with trastuzumab, an anti HER-2 antibody, has sub- ml rabbit anti-Wnt3a antibody (#2391; Cell Signaling stantially increased the overall survival rate of patients Technology, Massachusetts, USA) at 4 °C overnight and it with HER2-overexpressing cancers; however, trastu- was called Wnt3a-depletion medium. zumab resistance develops in most patients within a year [11–13]. Although new agents have been investigated to Trastuzumab-resistant gastric cancer cell lines delay the onset of resistance, the duration of response to HER-2 expression was detected using western blotting trastuzumab is limited by acquired resistance [14]. Thus, in all seven gastric cancer cell lines, including MKN45, it is necessary to characterize the resistance mechanism MKN74, SNU216, SNU484, NCI-N87, and AGS. SNU216 of trastuzumab to provide alternative treatment options and NCI-N87 cells exhibited the highest levels of HER-2 for patients that will inevitably develop resistance to the expression (Figure S1a). To simulate the in vivo mode of drug. resistance, we exposed SNU216 and NCI-N87 cells by Numerous factors, including loss of phosphatase and stepwise exposure to increasing doses of trastuzumab the tensin homolog gene (PTEN) function [15], muta- for over a year. Trastuzumab (12 µ g/ml) was added for tion of the phosphatidylinositide 3-kinase (PI3K)/AKT 48  h during the mitotic phase, and then the cells were pathway [16], upregulation of insulin-like growth factor transferred into drug-free culture medium until the receptor (IGFR) and hetero-dimerization of IGFR/HER- next mitotic phase (around 10 days). We continued this 20 [17], and accumulation of truncated HER-2 receptors process while observing cell death every day, changing (p95HER-2) have been found to be involved in trastu- to fresh complete culture medium, and performing the zumab resistance [18]. Previous studies suggested that MTT assay regularly. This process was continued until prolonged treatment of tumor cells with chemotherapeu- the concentration of trastuzumab in the medium reached tic drugs in vitro enriched the cell population with cancer 5000 µ g/ml after 360 days. In the NCI-N87 cell line, we stem cell (CSC) characteristics, such as high clonogenic- could successfully sub-cultured NCI-N87 cells, which ity, expression of stemness-related genes, self-renewal grew steadily in a medium containing trastuzumab, earn- ability, and resistance to chemotherapy [19]. Through ing the name NCI-N87R. The cells whose final resistance epithelial to mesenchymal transition (EMT), CSCs can was confirmed were completed around 30 passages, and emerge from differentiated cancer cells. The significance the numerical value was constantly monitored. How- of EMT in metastasis, tumor invasion, and drug resis- ever, SNU216 cell growths were not inhibited at all, and tance has become apparent [20, 21]. Moreover, EMT-like cell viability was not decreased even in trastuzumab transition is known to be regulated by EMT signaling (5000  µg/ml)-containing medium. As a result, we failed pathways, such as Wnt, Notch, and Hedgehog [16]. to induce the resistant cell line from SNU216 cells in this In a previous study, we demonstrated that HER-2 over- study, and it was assumed that they already had trastu- expressing gastric cancer cells exhibit stemness and an zumab-resistance. We observed the trastuzumab resis- EMT-like phenotype, which is mediated by the Wnt/β- tance of NCI-N87R cells, as revealed by the cell viability catenin signaling pathway [22]. Therefore, we focused on assay, while inhibition of trastuzumab on cell viability the Wnt signaling pathway and its influence on the phe - was seen to increase in a dose-dependent manner in notypes and behaviors of trastuzumab-resistant gastric NCI-N87 cells (Figure S1b). cancer cells. This study aimed to elucidate the mecha - nisms underlying trastuzumab resistance in HER2-over- expressing gastric cancer. Kim et al. BMC Cancer (2023) 23:922 Page 3 of 8 Cell viability assay with wash buffer (10 mM Tris-HCl, 70 mM NaCl, and At a density of 3.0-7.5 × 10 cells per well, cells were 0.05% Tween 20). The membrane was then washed thrice seeded in 96-well plates, incubated overnight at 37  °C, with wash buffer for 10 min, and ECL (Amersham Biosci - and then exposed to various concentrations of trastu- ences, GE Healthcare, Arlington Heights, IL, USA) was zumab for 72  h. Each well received a 50  µl aliquot of used for detection. 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) solution (Sigma-Aldrich, St. Louis, Immunofluorescence staining of E-Cadherin and β-Catenin MO, USA), and incubation was carried out at 37  °C for Rabbit monoclonal antibodies against E-cadherin an additional 4  h. After removing the medium, 150  µl (1:1,000; #sc-7870; Santa Cruz Biotechnology, Dallas, TX, of dimethyl sulfoxide (DMSO) was added to each well USA) and β-catenin (1:50; #sc-7199; Santa Cruz Biotech- and mixed. A VersaMax microplate reader (Molecular nology) were used to label the NCI-N87 and NCI-N87R Devices, Sunnyvale, CA, USA) was used to measure the cells. We observed the cells under a laser-scanning con- absorbance at 540 nm. focal microscope after we stained the nuclei with 1 g/mL DAPI (Sigma-Aldrich, St. Louis, MO, USA) (LSM 780; Spheroid colony formation assay ZEISS, Oberkochen, Germany). RPMI 1640 serum-free medium, 20 ng/ml human recom- binant basic fibroblast growth factor (Invitrogen), 20 ng/ Luciferase assay ml human recombinant epidermal growth factor (Invi- NCI-N87 and NCI-N87R cells were transfected with trogen, Carlsbad, CA, USA), and supplements N2 and pTA-Luc and TCF/LEF luciferase reporter vectors (Pro- B-27 were added to the trypsin-EDTA-isolated NCI-N87 mega, Madison, WI, USA). We co-transfected NCI-N87 and NCI-N87R cells before they were seeded in each and NCI-N87R cells with the TopFlash firefly luciferase well of an ultralow-attachment 96-well plate (Corning reporter vector and pRL-SV40-Renilla luciferase vector Life Sciences, Acton, MA, USA). Every 4 days, 20 µl of (Promega). Additionally, we incubated the cells for 72  h the medium was replaced. Each well was examined under to detect the Wnt signaling pathway activity. The dual- a light microscope after 5, 14, and 21 days, and the size luciferase reporter method (Promega) was used to deter- of the spheroidal cells was measured and compared with mine the relative luciferase activity. that of the wild-type cells. Organoid culture Western blotting analysis After the study was approved by the ethical committee Whole cells were centrifuged at 15,000  rpm for 10  min (IRB 3-2018-0209), clinical samples for organoid estab- at 4  °C after lysis in a RIPA lysis buffer for over 45  min. lishment and biological analyses were obtained from The supernatant protein concentration was determined patients at Gangnam Severance Hospital with informed using a Bradford assay kit (Bio-Rad Laboratories, Her- consent. Gastric cancer specimens were collected via sur- cules, CA, USA) or a BCA protein assay kit (Thermo gical resection or biopsy. Surgical specimens were washed scientific, Rockford, MR, USA). Thirty micrograms of with phosphate-buffered saline (PBS) before being cut denatured protein from each sample were then trans- into 1-mm fragments. The fragments were digested ferred to a PVDF membrane (Millipore, Billerica, MA, with collagenase I (Sigma-Aldrich, St. Louis, MO, USA) USA) after being separated on a 10% SDS-PAGE gel. at 37  °C for 1  h, and undigested pellets were separated The membranes were blocked with 5% skim milk or BSA by pressing with a plastic stick. To inactivate the diges- for 1  h at RT. Next, the membrane was incubated over- tive enzymes, the collected epithelia were washed with night at 4 °C with rabbit anti-polycomb complex protein PBS that was supplemented with 1% bovine serum albu- BMI-1 (BMI1) (1: 500; #ab135713; Abcam, Cambridge, min before performing the plating. For the basal culture UK), rabbit anti-HER2 (1: 1,000; #2165; Cell Signaling medium, advanced Dulbecco’s modified Eagle’s medium/ Technology, Danvers, MA, USA), rabbit anti-Snail (1: F12 was supplemented with 10 mM HEPES, antibiotic/ 500; #3895; Cell Signaling Technology, Massachusetts, antimycotic, 1 × B27 supplement (Thermo Fisher Sci - USA), rabbit anti-octamer-binding transcription factor 4 entific), and 2 mM GlutaMAX. The niche factors were (Oct4) (1: 1,000; #2750; Cell Signaling Technology, Mas- added to the basal culture medium to create a complete sachusetts, USA), rabbit anti Oct4a (1: 500; #2890; Cell medium. The organoids were maintained in an incuba - Signaling Technology, Massachusetts, USA), and rabbit tor at 37 °C with 5% CO2 and the medium was changed anti-GAPDH (1: 2,000; #2118; Cell Signaling Technology, every 3–4 days. Massachusetts, USA) primary antibodies. The membrane was incubated for 1  h at RT with HRP-conjugated anti- Statistical analysis rabbit IgG (1:5,000; #7074; Cell Signaling Technology, All statistical analyses were conducted using SPSS ver- Massachusetts, USA) secondary antibodies after washing sion 26.0. One-way analysis of variance (ANOVA) was Kim et al. BMC Cancer (2023) 23:922 Page 4 of 8 used to determine the difference between the subgroups. Trastuzumab-resistant gastric cancer cells exhibit A P value of less than 0.05 meant that the difference was increased activity of wnt signaling pathway statistically significant. As trastuzumab-resistant gastric cancer cells demon- strated EMT-like phenotype changes, we analyzed the Results activity of the Wnt signaling pathway, which is known Trastuzumab-resistant gastric cancer cells exhibit stemness to play a crucial role in EMT. The TCF/LEF reporter kit and EMT-like phenotypes was used to assess the Wnt signaling pathway activity. To evaluate the stemness of trastuzumab-resistant gastric NCI-N87R cells showed significantly higher activity of cancer cells, parental NCI-N87 and NCI-N87R cells were the Wnt signaling pathway than parental cells (Fig.  2a). cultured in a suspension for 21 days. Both cell types pro- Likewise, immunofluorescence staining showed that duced nonadherent spherical colonies known as spheres. β-catenin was significantly upregulated in NCI-N87R Compared with NCI-N87 cells, NCI-N87R cells had sig- cells compared with parental cells (Fig. 2b). nificantly larger sphere size with a higher cell count of spheres larger than 50 μm (Fig. 1a). Loss of expression of Gastric cancer cells incubated in Wnt3a-conditioned the epithelial marker E-cadherin is a hallmark of EMT. medium exhibit increased activity of wnt signaling E-cadherin was significantly downregulated in NCI- pathway N87R cells, as confirmed by immunofluorescence stain - The activity of the Wnt signaling pathway, which plays ing. Stem cell markers, including CD44s, CD54, BMI1, an important role in EMT, was found to be increased in OCT4, Vimentin, and Snail, were significantly upregu - trastuzumab-resistant gastric cancer cells. Therefore, we lated in NCI-N87R cells compared with parental cells, investigated whether the Wnt signaling pathway could be as determined by western blot analysis (Fig.  1b). These activated when parental gastric cancer cells were incu- results suggest that trastuzumab-resistant gastric cancer bated in the Wnt3a-conditioned medium. NCI-N87 cells cells exhibit stemness and overlapping characteristics incubated in Wnt3a-conditioned medium (NCI-N87_ with EMT cells. WNT) showed significantly increased activity of the Wnt signaling pathway (Fig.  3a). Immunofluorescence stain - ing revealed that NCI-N87_WNT cells had markedly higher levels of β-catenin expression than NCI-N87 cells (Fig.  3b). Additionally, E-cadherin was downregulated Fig. 1 Stemness and EMT-like phenotypes of trastuzumab-resistant gastric cancer. (a) NCI-N87R cells had significantly larger sphere size and comprised higher cell counts of spheres larger than 50 μm compared with NCI-N87 cells. (b) E-cadherin was significantly downregulated in NCI-N87R cells, as confirmed by immunofluorescence, compared with parental cells. Stem cell markers including CD44s, CD54, BMI1, Oct4, Vimentin and Snail were significantly upregulated in NCI-N87R cells compared with parental cells, according to Western blot analysis. * P < 0.05, **P < 0.01, ***P < 0.005 Kim et al. BMC Cancer (2023) 23:922 Page 5 of 8 Fig. 2 Increased activity of Wnt signaling pathway of tastuzumab-resistant gastric cancer cells. (a) NCI-N87R cells showed significantly higher activity of the Wnt signaling pathway compared with that of parental cells. (b) β-catenin was significantly upregulated in NCI-N87R cells compared with parental cells, as observed on immunofluorescence staining. P < 0.05, **P < 0.01, ***P < 0.005 Fig. 3 Increased activity of Wnt signaling pathway of gastric cancer cells incubated in Wnt3a-conditioned medium. (a) NCI-N87_WNT cells showed significantly increased activity of the Wnt signaling pathway. (b) Immunofluorescence staining revealed that NCI-N87_WNT cells had markedly higher levels of β-catenin expression than NCI-N87 cells. (c) E-cadherin was downregulated in NCI-N87_WNT cells, and this was similar in NCI-N87R cells. *P < 0.05, **P < 0.01, ***P < 0.005 in NCI-N87_WNT cells as well as in NCI-N87R cells Wnt signaling pathway affected trastuzumab resistance of (Fig. 3c). gastric cancer in patients-derived organoids Next, we investigated whether the Wnt signaling path- Gastric cancer cells incubated in Wnt3a-conditioned way affected trastuzumab resistance on cell viability in medium acquired trastuzumab resistance a patient-derived organoid. “GC032” and “GC098” are We compared the trastuzumab resistance ability of each the names of organoid cells derived from gastric cancer cell line using a cell viability assay. NCI-N87_WNT cells patients and cultured in complete medium. “GC032_(-) showed lower inhibition of cell proliferation than paren- WNT” and “GC098_(-)WNT” indicate that “GC032” and tal cells (Fig. 4). “GC098” incubated in Wnt3a-depletion medium. We Kim et al. BMC Cancer (2023) 23:922 Page 6 of 8 In the current study, trastuzumab-resistant cells were obtained in vitro from the human gastric cancer cell lines NCI-N87 through repeated, dose-escalating trastu- zumab treatment. Trastuzumab-resistant gastric cancer cells showed higher levels of stemness and EMT charac- teristics along with obvious acquisition of mesenchymal morphology, decreased levels of epithelial markers, and increased levels of mesenchymal markers. Self-renewal (forming spheres), increased clonogenicity, and tumori- genicity were also observed in trastuzumab-resistant gastric cancer cells. EMT can trigger reversion to a CSC. CSCs, which can initiate tumorigenesis and have high metastatic potential, frequently develop resistance to chemotherapeutic agents [20]. The combination of Fig. 4 Acquired trastuzumab resistance of gastric cancer cells in- stemness and EMT is an independent predictor of out- cubated in Wnt3a-conditioned medium. NCI-N87_WNT cells showed comes in patients with gastric cancer [23]. It was recently lower inhibition of cell proliferation than parental cells shown that ectopic expression of the embryonic stem measured the cell viability of each organoid cell by expos- cells transcription factor, NANOGP8, in gastric cancer ing them to escalating doses of trastuzumab. Organoid cells, promotes sphere formation and chemo-resistance cells incubated in Wnt3a-depletion medium were more by up-regulating EMT inducers and CSCs markers [24]. susceptible to dose-dependent inhibition of cell viabil- Furthermore, drug resistance is substantially correlated ity by trastuzumab than organoids cultured in complete with the expression of LGR5 and EMT-related genes in medium (Fig. 5). gastric cancer sphere cells [25]. These findings suggest that prolonged trastuzumab treatment induces stemness Discussion and EMT-like phenotype in gastric cancer cells, leading Trastuzumab, one of the most effective anti-HER2 anti - to trastuzumab resistance. bodies for breast and gastric cancers, has been used in Through EMT induction, pathophysiological condi - clinical therapy for a long time; however, the emergence tions, such as tissue damage or tumorigenesis, can cause of resistance is a significant barrier to trastuzumab-based differentiated cells to develop a multipotent stem cell-like treatment for HER2-overexpressing breast and gastric phenotype. This process is similar to developmentally cancers. Although numerous mechanisms of trastu- regulated EMT signaling pathways, such as Wnt, Notch, zumab resistance have been suggested in association and Hedgehog, which are responsible for both normal with breast cancer, it is unclear whether the same mecha- and CSC renewal and maintenance [26, 27]. Wnt is a nism applies for gastric cancer. Therefore, it is crucial to highly conserved signaling pathway that regulates embry- understand the mechanisms and identify the phenotype onic and organ development as well as the progression of trastuzumab resistance in gastric cancer to develop of various types of human cancers, such as breast cancer, novel therapeutic approaches. ovarian cancer, colorectal cancer, and prostate cancer Fig. 5 Trastuzumab resistance of gastric cancer in patients-derived organoids affected by Wnt signaling pathway. Gastric cancer organoid cells incubated in Wnt3a-depletion medium were more susceptible to dose-dependent inhibition of cell viability by trastuzumab than in complete medium Kim et al. BMC Cancer (2023) 23:922 Page 7 of 8 [28]. Recent genome-wide sequencing and gene expres- [21]. We conclude that the Wnt signaling pathway may be sion profile analyses have revealed that Wnt signaling is a target for trastuzumab-resistant gastric cancer, and that primarily involved in cancer proliferation and metastasis. Wnt signaling pathway inhibitors combined with trastu- Recent studies have revealed that Wnt signaling is essen- zumab may provide a promising treatment strategy for tial for breast cancer immune microenvironment regu- patients with trastuzumab-refractory gastric cancer. Fur- lation, stemness maintenance, therapeutic resistance, ther research is required to understand the trastuzumab- and phenotype shaping [29–31]. Wu et al. showed that resistant mechanism for an individualized and precise Wnt3 overexpression in breast cancer cells resistant to treatment of gastric cancer. trastuzumab activates the Wnt signaling pathway, which induces transactivation of EGFR and promotes EMT-like Supplementary Information The online version contains supplementary material available at https://doi. transition [16]. The EMT-like transition in cancer cells org/10.1186/s12885-023-11447-4. may promote tumor invasion, metastases, and drug resis- tance. Data from our current study indicate that trastu- Supplementary Material 1 zumab-resistant gastric cancer cells exhibit increased Supplementary Material 2 activity of the Wnt signaling pathway. Additionally gastric cancer cells incubated in Wnt3a-conditioned medium Acknowledgements exhibit increased activity of Wnt signaling pathway and Not applicable. trastuzumab resistance. We also investigated how the Authors’ contributions Wnt signaling pathway affected trastuzumab resistance All authors contributed to the study conception and design. Material on cell viability in a patient-derived preclinical model. preparation, data collection and analysis were performed by Yuna Kim, Yoo Organoid cells incubated in Wnt3a-depletion medium Jin Bae, Jie-Hyun Kim. The first draft of the manuscript was written by Yuna Kim and Yoo Jin Bae, and all authors commented on previous versions of the were more susceptible to dose-dependent inhibition of manuscript. All authors read and approved the final manuscript. cell viability by trastuzumab than the parental cells. In several recent studies, it was importantly noted that Funding This work was supported by the Basic Science Research Program through EMT-related signaling interacts functionally with the the National Research Foundation of Korea (NRF), funded by the Ministry autophagy pathway, which is intricately linked to the fate of Education, Science, and Technology (2021R1A2C2011296). This study of cancer cells [32]. Interestingly, autophagy also exhib- was supported by a faculty research grant from Yonsei University College of Medicine (6-2018-0077, 6-2020-0233). its a dual effect on EMT, depending on the setting, either activating or inhibiting it. Through modulating autoph - Data Availability agy signaling pathways, such as integrin, NF-κB, Wnt The data that support the findings of this study are available from the corresponding author upon reasonable request. and TGF-β, the EMT process has a dramatic effect on autophagy modulation [33]. Given the complexity of this Declarations interaction, elucidating the mechanisms of their mutual regulation is challenging, but have clinical benefits in Competing interests cancer treatment. The authors declare no competing interests. This study has limitations that need to be addressed. Ethics approval and consent to participate First, we could obtain only one resistant cell line of NCI- The protocol of this original study was in accordance with the ethical N87R. When we exposed SNU216 and NCI-N87 cells to standards of the responsible committee on human experimentation and with the Helsinki Declaration. After the study was approved by the Institutional trastuzumab, SNU 216 cells were unable to produce resis- Review Board of Gangnam Severance Hospital (IRB 3-2018-0209), clinical tant cells and it was consequently assumed that the SNU samples for organoid establishment and biological analyses were obtained 216 cell line already exhibited trastuzumab-resistance. from patients at Gangnam Severance Hospital with informed consent. In addition, knock-down or knock-out of Wnt3a study is Patient consent for publication required to ascertain whether the Wnt/β-catenin path- Not applicable. way is critical for maintenance of trastuzumab resistance. Our study revealed that trastuzumab-resistant gastric Received: 17 May 2023 / Accepted: 25 September 2023 cancer cells exhibit EMT-like phenotype by promoting the Wnt signaling pathway. 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Wnt/β-catenin pathway is a key signaling pathway to trastuzumab resistance in gastric cancer cells

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Abstract

Background Trastuzumab is the only approved target agent for the first-line treatment of human epidermal growth factor receptor-2 (HER-2) positive gastric cancer; however, trastuzumab resistance is a major problem in clinical practice. To comprehend the mechanism of trastuzumab resistance, we focused on the Wnt/β-catenin signaling pathway and its influence on the phenotypes and behavior of trastuzumab-resistant gastric cancer cells. Methods Trastuzumab-resistant NCI-N87R cells were established in vitro from the human gastric cancer cell line NCI-N87 by dose-escalating repeated trastuzumab treatment. We investigated the phenotypes of NCI-N87R cells, including Wnt signaling pathway activity. Gastric cancer organoid cells were incubated with complete medium and Wnt3a-depletion medium, and their resistance to trastuzumab was compared. Results NCI-N87R exhibited stemness and epithelial-mesenchymal transition (EMT )-like phenotypes, along with decreased levels of the epithelial marker E-cadherin and increased levels of the mesenchymal markers Vimentin and Snail along with an increased Wnt signaling pathway activity. When gastric cancer cells were incubated in Wnt3a- conditioned medium. Wnt signaling pathway activity and resistance to trastuzumab increased. Gastric cancer patient- derived organoids incubated in Wnt3a-depletion medium were more susceptible to dose-dependent inhibition of cell viability by trastuzumab than those incubated in complete medium. Conclusions Trastuzumab-resistant gastric cancer cells exhibited EMT-like phenotype, and trastuzumab resistance was promoted by the Wnt/β-catenin signaling pathway. The Wnt/β-catenin pathway is a key signaling pathway for trastuzumab resistance in gastric cancer cells. Keywords Wnt, Gastric cancer, Trastuzumab, Resistance, Epithelial to mesenchymal transition † Department of Pathology, Yonsei University College of Medicine, Yuna Kim and Yoo Jin Bae These two authors contributed equally Seoul 03722, Korea to this work. Department of Pathology, Gangnam Severance Hospital, Yonsei *Correspondence: University College of Medicine, Seoul 06229, Korea Jie-Hyun Kim Department of Internal Medicine, Division of Gastroenterology, Yonsei otilia94@yuhs.ac University College of Medicine, Seoul 03722, Korea Department of Internal Medicine, Division of Gastroenterology, Gangnam Severance Hospital, Yonsei University College of Medicine, 20, Eonju-ro 63-gil, Gangnam-gu, Seoul 06229, Korea © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Kim et al. BMC Cancer (2023) 23:922 Page 2 of 8 Introduction Materials and methods Gastric cancer ranks fifth in incidence and fourth in Gastric cancer cell line and culture mortality globally, with an estimate of one million new Gastric cancer cell lines including MKN45, MKN74, cases and 769,000 deaths reported in 2020 [1]. Human SNU216, SNU484, NCI-N87, and AGS were obtained epidermal growth factor receptor-2 (HER-2), a member from and authenticated by the Korean Cell Line Bank of the epidermal growth factor receptor (EGFR) family, with STR profiling. All cells were cultured in the RPMI is an important treatment target for gastric cancer [2]. 1640 medium that had 10% FBS added to it at 37 °C and Amplification of the HER2 gene is observed in approxi - 5% CO in a humid incubator. mately 20% of patients with gastric cancer [3, 4]. Previ- ous studies have suggested that HER-2 overexpression Preparation of conditioned medium is positively associated with cancer cell proliferation, Wnt3a-conditioned medium was harvested from Wnt3a malignancy, metastasis, and unfavorable outcomes [2, expressing L-cells (CRL-2647; American Type Culture 5, 6]. HER-2 overexpression has also been observed in Collection, Manassas, VA) according to the manufac- other solid tumors, including biliary tract, colorectal, turer’s instructions. Wnt3a-conditioned medium was non-small-cell lung, and bladder cancers [7–10]. Treat- depleted of the Wnt3a protein by incubation with 4  µg/ ment with trastuzumab, an anti HER-2 antibody, has sub- ml rabbit anti-Wnt3a antibody (#2391; Cell Signaling stantially increased the overall survival rate of patients Technology, Massachusetts, USA) at 4 °C overnight and it with HER2-overexpressing cancers; however, trastu- was called Wnt3a-depletion medium. zumab resistance develops in most patients within a year [11–13]. Although new agents have been investigated to Trastuzumab-resistant gastric cancer cell lines delay the onset of resistance, the duration of response to HER-2 expression was detected using western blotting trastuzumab is limited by acquired resistance [14]. Thus, in all seven gastric cancer cell lines, including MKN45, it is necessary to characterize the resistance mechanism MKN74, SNU216, SNU484, NCI-N87, and AGS. SNU216 of trastuzumab to provide alternative treatment options and NCI-N87 cells exhibited the highest levels of HER-2 for patients that will inevitably develop resistance to the expression (Figure S1a). To simulate the in vivo mode of drug. resistance, we exposed SNU216 and NCI-N87 cells by Numerous factors, including loss of phosphatase and stepwise exposure to increasing doses of trastuzumab the tensin homolog gene (PTEN) function [15], muta- for over a year. Trastuzumab (12 µ g/ml) was added for tion of the phosphatidylinositide 3-kinase (PI3K)/AKT 48  h during the mitotic phase, and then the cells were pathway [16], upregulation of insulin-like growth factor transferred into drug-free culture medium until the receptor (IGFR) and hetero-dimerization of IGFR/HER- next mitotic phase (around 10 days). We continued this 20 [17], and accumulation of truncated HER-2 receptors process while observing cell death every day, changing (p95HER-2) have been found to be involved in trastu- to fresh complete culture medium, and performing the zumab resistance [18]. Previous studies suggested that MTT assay regularly. This process was continued until prolonged treatment of tumor cells with chemotherapeu- the concentration of trastuzumab in the medium reached tic drugs in vitro enriched the cell population with cancer 5000 µ g/ml after 360 days. In the NCI-N87 cell line, we stem cell (CSC) characteristics, such as high clonogenic- could successfully sub-cultured NCI-N87 cells, which ity, expression of stemness-related genes, self-renewal grew steadily in a medium containing trastuzumab, earn- ability, and resistance to chemotherapy [19]. Through ing the name NCI-N87R. The cells whose final resistance epithelial to mesenchymal transition (EMT), CSCs can was confirmed were completed around 30 passages, and emerge from differentiated cancer cells. The significance the numerical value was constantly monitored. How- of EMT in metastasis, tumor invasion, and drug resis- ever, SNU216 cell growths were not inhibited at all, and tance has become apparent [20, 21]. Moreover, EMT-like cell viability was not decreased even in trastuzumab transition is known to be regulated by EMT signaling (5000  µg/ml)-containing medium. As a result, we failed pathways, such as Wnt, Notch, and Hedgehog [16]. to induce the resistant cell line from SNU216 cells in this In a previous study, we demonstrated that HER-2 over- study, and it was assumed that they already had trastu- expressing gastric cancer cells exhibit stemness and an zumab-resistance. We observed the trastuzumab resis- EMT-like phenotype, which is mediated by the Wnt/β- tance of NCI-N87R cells, as revealed by the cell viability catenin signaling pathway [22]. Therefore, we focused on assay, while inhibition of trastuzumab on cell viability the Wnt signaling pathway and its influence on the phe - was seen to increase in a dose-dependent manner in notypes and behaviors of trastuzumab-resistant gastric NCI-N87 cells (Figure S1b). cancer cells. This study aimed to elucidate the mecha - nisms underlying trastuzumab resistance in HER2-over- expressing gastric cancer. Kim et al. BMC Cancer (2023) 23:922 Page 3 of 8 Cell viability assay with wash buffer (10 mM Tris-HCl, 70 mM NaCl, and At a density of 3.0-7.5 × 10 cells per well, cells were 0.05% Tween 20). The membrane was then washed thrice seeded in 96-well plates, incubated overnight at 37  °C, with wash buffer for 10 min, and ECL (Amersham Biosci - and then exposed to various concentrations of trastu- ences, GE Healthcare, Arlington Heights, IL, USA) was zumab for 72  h. Each well received a 50  µl aliquot of used for detection. 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) solution (Sigma-Aldrich, St. Louis, Immunofluorescence staining of E-Cadherin and β-Catenin MO, USA), and incubation was carried out at 37  °C for Rabbit monoclonal antibodies against E-cadherin an additional 4  h. After removing the medium, 150  µl (1:1,000; #sc-7870; Santa Cruz Biotechnology, Dallas, TX, of dimethyl sulfoxide (DMSO) was added to each well USA) and β-catenin (1:50; #sc-7199; Santa Cruz Biotech- and mixed. A VersaMax microplate reader (Molecular nology) were used to label the NCI-N87 and NCI-N87R Devices, Sunnyvale, CA, USA) was used to measure the cells. We observed the cells under a laser-scanning con- absorbance at 540 nm. focal microscope after we stained the nuclei with 1 g/mL DAPI (Sigma-Aldrich, St. Louis, MO, USA) (LSM 780; Spheroid colony formation assay ZEISS, Oberkochen, Germany). RPMI 1640 serum-free medium, 20 ng/ml human recom- binant basic fibroblast growth factor (Invitrogen), 20 ng/ Luciferase assay ml human recombinant epidermal growth factor (Invi- NCI-N87 and NCI-N87R cells were transfected with trogen, Carlsbad, CA, USA), and supplements N2 and pTA-Luc and TCF/LEF luciferase reporter vectors (Pro- B-27 were added to the trypsin-EDTA-isolated NCI-N87 mega, Madison, WI, USA). We co-transfected NCI-N87 and NCI-N87R cells before they were seeded in each and NCI-N87R cells with the TopFlash firefly luciferase well of an ultralow-attachment 96-well plate (Corning reporter vector and pRL-SV40-Renilla luciferase vector Life Sciences, Acton, MA, USA). Every 4 days, 20 µl of (Promega). Additionally, we incubated the cells for 72  h the medium was replaced. Each well was examined under to detect the Wnt signaling pathway activity. The dual- a light microscope after 5, 14, and 21 days, and the size luciferase reporter method (Promega) was used to deter- of the spheroidal cells was measured and compared with mine the relative luciferase activity. that of the wild-type cells. Organoid culture Western blotting analysis After the study was approved by the ethical committee Whole cells were centrifuged at 15,000  rpm for 10  min (IRB 3-2018-0209), clinical samples for organoid estab- at 4  °C after lysis in a RIPA lysis buffer for over 45  min. lishment and biological analyses were obtained from The supernatant protein concentration was determined patients at Gangnam Severance Hospital with informed using a Bradford assay kit (Bio-Rad Laboratories, Her- consent. Gastric cancer specimens were collected via sur- cules, CA, USA) or a BCA protein assay kit (Thermo gical resection or biopsy. Surgical specimens were washed scientific, Rockford, MR, USA). Thirty micrograms of with phosphate-buffered saline (PBS) before being cut denatured protein from each sample were then trans- into 1-mm fragments. The fragments were digested ferred to a PVDF membrane (Millipore, Billerica, MA, with collagenase I (Sigma-Aldrich, St. Louis, MO, USA) USA) after being separated on a 10% SDS-PAGE gel. at 37  °C for 1  h, and undigested pellets were separated The membranes were blocked with 5% skim milk or BSA by pressing with a plastic stick. To inactivate the diges- for 1  h at RT. Next, the membrane was incubated over- tive enzymes, the collected epithelia were washed with night at 4 °C with rabbit anti-polycomb complex protein PBS that was supplemented with 1% bovine serum albu- BMI-1 (BMI1) (1: 500; #ab135713; Abcam, Cambridge, min before performing the plating. For the basal culture UK), rabbit anti-HER2 (1: 1,000; #2165; Cell Signaling medium, advanced Dulbecco’s modified Eagle’s medium/ Technology, Danvers, MA, USA), rabbit anti-Snail (1: F12 was supplemented with 10 mM HEPES, antibiotic/ 500; #3895; Cell Signaling Technology, Massachusetts, antimycotic, 1 × B27 supplement (Thermo Fisher Sci - USA), rabbit anti-octamer-binding transcription factor 4 entific), and 2 mM GlutaMAX. The niche factors were (Oct4) (1: 1,000; #2750; Cell Signaling Technology, Mas- added to the basal culture medium to create a complete sachusetts, USA), rabbit anti Oct4a (1: 500; #2890; Cell medium. The organoids were maintained in an incuba - Signaling Technology, Massachusetts, USA), and rabbit tor at 37 °C with 5% CO2 and the medium was changed anti-GAPDH (1: 2,000; #2118; Cell Signaling Technology, every 3–4 days. Massachusetts, USA) primary antibodies. The membrane was incubated for 1  h at RT with HRP-conjugated anti- Statistical analysis rabbit IgG (1:5,000; #7074; Cell Signaling Technology, All statistical analyses were conducted using SPSS ver- Massachusetts, USA) secondary antibodies after washing sion 26.0. One-way analysis of variance (ANOVA) was Kim et al. BMC Cancer (2023) 23:922 Page 4 of 8 used to determine the difference between the subgroups. Trastuzumab-resistant gastric cancer cells exhibit A P value of less than 0.05 meant that the difference was increased activity of wnt signaling pathway statistically significant. As trastuzumab-resistant gastric cancer cells demon- strated EMT-like phenotype changes, we analyzed the Results activity of the Wnt signaling pathway, which is known Trastuzumab-resistant gastric cancer cells exhibit stemness to play a crucial role in EMT. The TCF/LEF reporter kit and EMT-like phenotypes was used to assess the Wnt signaling pathway activity. To evaluate the stemness of trastuzumab-resistant gastric NCI-N87R cells showed significantly higher activity of cancer cells, parental NCI-N87 and NCI-N87R cells were the Wnt signaling pathway than parental cells (Fig.  2a). cultured in a suspension for 21 days. Both cell types pro- Likewise, immunofluorescence staining showed that duced nonadherent spherical colonies known as spheres. β-catenin was significantly upregulated in NCI-N87R Compared with NCI-N87 cells, NCI-N87R cells had sig- cells compared with parental cells (Fig. 2b). nificantly larger sphere size with a higher cell count of spheres larger than 50 μm (Fig. 1a). Loss of expression of Gastric cancer cells incubated in Wnt3a-conditioned the epithelial marker E-cadherin is a hallmark of EMT. medium exhibit increased activity of wnt signaling E-cadherin was significantly downregulated in NCI- pathway N87R cells, as confirmed by immunofluorescence stain - The activity of the Wnt signaling pathway, which plays ing. Stem cell markers, including CD44s, CD54, BMI1, an important role in EMT, was found to be increased in OCT4, Vimentin, and Snail, were significantly upregu - trastuzumab-resistant gastric cancer cells. Therefore, we lated in NCI-N87R cells compared with parental cells, investigated whether the Wnt signaling pathway could be as determined by western blot analysis (Fig.  1b). These activated when parental gastric cancer cells were incu- results suggest that trastuzumab-resistant gastric cancer bated in the Wnt3a-conditioned medium. NCI-N87 cells cells exhibit stemness and overlapping characteristics incubated in Wnt3a-conditioned medium (NCI-N87_ with EMT cells. WNT) showed significantly increased activity of the Wnt signaling pathway (Fig.  3a). Immunofluorescence stain - ing revealed that NCI-N87_WNT cells had markedly higher levels of β-catenin expression than NCI-N87 cells (Fig.  3b). Additionally, E-cadherin was downregulated Fig. 1 Stemness and EMT-like phenotypes of trastuzumab-resistant gastric cancer. (a) NCI-N87R cells had significantly larger sphere size and comprised higher cell counts of spheres larger than 50 μm compared with NCI-N87 cells. (b) E-cadherin was significantly downregulated in NCI-N87R cells, as confirmed by immunofluorescence, compared with parental cells. Stem cell markers including CD44s, CD54, BMI1, Oct4, Vimentin and Snail were significantly upregulated in NCI-N87R cells compared with parental cells, according to Western blot analysis. * P < 0.05, **P < 0.01, ***P < 0.005 Kim et al. BMC Cancer (2023) 23:922 Page 5 of 8 Fig. 2 Increased activity of Wnt signaling pathway of tastuzumab-resistant gastric cancer cells. (a) NCI-N87R cells showed significantly higher activity of the Wnt signaling pathway compared with that of parental cells. (b) β-catenin was significantly upregulated in NCI-N87R cells compared with parental cells, as observed on immunofluorescence staining. P < 0.05, **P < 0.01, ***P < 0.005 Fig. 3 Increased activity of Wnt signaling pathway of gastric cancer cells incubated in Wnt3a-conditioned medium. (a) NCI-N87_WNT cells showed significantly increased activity of the Wnt signaling pathway. (b) Immunofluorescence staining revealed that NCI-N87_WNT cells had markedly higher levels of β-catenin expression than NCI-N87 cells. (c) E-cadherin was downregulated in NCI-N87_WNT cells, and this was similar in NCI-N87R cells. *P < 0.05, **P < 0.01, ***P < 0.005 in NCI-N87_WNT cells as well as in NCI-N87R cells Wnt signaling pathway affected trastuzumab resistance of (Fig. 3c). gastric cancer in patients-derived organoids Next, we investigated whether the Wnt signaling path- Gastric cancer cells incubated in Wnt3a-conditioned way affected trastuzumab resistance on cell viability in medium acquired trastuzumab resistance a patient-derived organoid. “GC032” and “GC098” are We compared the trastuzumab resistance ability of each the names of organoid cells derived from gastric cancer cell line using a cell viability assay. NCI-N87_WNT cells patients and cultured in complete medium. “GC032_(-) showed lower inhibition of cell proliferation than paren- WNT” and “GC098_(-)WNT” indicate that “GC032” and tal cells (Fig. 4). “GC098” incubated in Wnt3a-depletion medium. We Kim et al. BMC Cancer (2023) 23:922 Page 6 of 8 In the current study, trastuzumab-resistant cells were obtained in vitro from the human gastric cancer cell lines NCI-N87 through repeated, dose-escalating trastu- zumab treatment. Trastuzumab-resistant gastric cancer cells showed higher levels of stemness and EMT charac- teristics along with obvious acquisition of mesenchymal morphology, decreased levels of epithelial markers, and increased levels of mesenchymal markers. Self-renewal (forming spheres), increased clonogenicity, and tumori- genicity were also observed in trastuzumab-resistant gastric cancer cells. EMT can trigger reversion to a CSC. CSCs, which can initiate tumorigenesis and have high metastatic potential, frequently develop resistance to chemotherapeutic agents [20]. The combination of Fig. 4 Acquired trastuzumab resistance of gastric cancer cells in- stemness and EMT is an independent predictor of out- cubated in Wnt3a-conditioned medium. NCI-N87_WNT cells showed comes in patients with gastric cancer [23]. It was recently lower inhibition of cell proliferation than parental cells shown that ectopic expression of the embryonic stem measured the cell viability of each organoid cell by expos- cells transcription factor, NANOGP8, in gastric cancer ing them to escalating doses of trastuzumab. Organoid cells, promotes sphere formation and chemo-resistance cells incubated in Wnt3a-depletion medium were more by up-regulating EMT inducers and CSCs markers [24]. susceptible to dose-dependent inhibition of cell viabil- Furthermore, drug resistance is substantially correlated ity by trastuzumab than organoids cultured in complete with the expression of LGR5 and EMT-related genes in medium (Fig. 5). gastric cancer sphere cells [25]. These findings suggest that prolonged trastuzumab treatment induces stemness Discussion and EMT-like phenotype in gastric cancer cells, leading Trastuzumab, one of the most effective anti-HER2 anti - to trastuzumab resistance. bodies for breast and gastric cancers, has been used in Through EMT induction, pathophysiological condi - clinical therapy for a long time; however, the emergence tions, such as tissue damage or tumorigenesis, can cause of resistance is a significant barrier to trastuzumab-based differentiated cells to develop a multipotent stem cell-like treatment for HER2-overexpressing breast and gastric phenotype. This process is similar to developmentally cancers. Although numerous mechanisms of trastu- regulated EMT signaling pathways, such as Wnt, Notch, zumab resistance have been suggested in association and Hedgehog, which are responsible for both normal with breast cancer, it is unclear whether the same mecha- and CSC renewal and maintenance [26, 27]. Wnt is a nism applies for gastric cancer. Therefore, it is crucial to highly conserved signaling pathway that regulates embry- understand the mechanisms and identify the phenotype onic and organ development as well as the progression of trastuzumab resistance in gastric cancer to develop of various types of human cancers, such as breast cancer, novel therapeutic approaches. ovarian cancer, colorectal cancer, and prostate cancer Fig. 5 Trastuzumab resistance of gastric cancer in patients-derived organoids affected by Wnt signaling pathway. Gastric cancer organoid cells incubated in Wnt3a-depletion medium were more susceptible to dose-dependent inhibition of cell viability by trastuzumab than in complete medium Kim et al. BMC Cancer (2023) 23:922 Page 7 of 8 [28]. Recent genome-wide sequencing and gene expres- [21]. We conclude that the Wnt signaling pathway may be sion profile analyses have revealed that Wnt signaling is a target for trastuzumab-resistant gastric cancer, and that primarily involved in cancer proliferation and metastasis. Wnt signaling pathway inhibitors combined with trastu- Recent studies have revealed that Wnt signaling is essen- zumab may provide a promising treatment strategy for tial for breast cancer immune microenvironment regu- patients with trastuzumab-refractory gastric cancer. Fur- lation, stemness maintenance, therapeutic resistance, ther research is required to understand the trastuzumab- and phenotype shaping [29–31]. Wu et al. showed that resistant mechanism for an individualized and precise Wnt3 overexpression in breast cancer cells resistant to treatment of gastric cancer. trastuzumab activates the Wnt signaling pathway, which induces transactivation of EGFR and promotes EMT-like Supplementary Information The online version contains supplementary material available at https://doi. transition [16]. The EMT-like transition in cancer cells org/10.1186/s12885-023-11447-4. may promote tumor invasion, metastases, and drug resis- tance. Data from our current study indicate that trastu- Supplementary Material 1 zumab-resistant gastric cancer cells exhibit increased Supplementary Material 2 activity of the Wnt signaling pathway. Additionally gastric cancer cells incubated in Wnt3a-conditioned medium Acknowledgements exhibit increased activity of Wnt signaling pathway and Not applicable. trastuzumab resistance. We also investigated how the Authors’ contributions Wnt signaling pathway affected trastuzumab resistance All authors contributed to the study conception and design. Material on cell viability in a patient-derived preclinical model. preparation, data collection and analysis were performed by Yuna Kim, Yoo Organoid cells incubated in Wnt3a-depletion medium Jin Bae, Jie-Hyun Kim. The first draft of the manuscript was written by Yuna Kim and Yoo Jin Bae, and all authors commented on previous versions of the were more susceptible to dose-dependent inhibition of manuscript. All authors read and approved the final manuscript. cell viability by trastuzumab than the parental cells. In several recent studies, it was importantly noted that Funding This work was supported by the Basic Science Research Program through EMT-related signaling interacts functionally with the the National Research Foundation of Korea (NRF), funded by the Ministry autophagy pathway, which is intricately linked to the fate of Education, Science, and Technology (2021R1A2C2011296). This study of cancer cells [32]. Interestingly, autophagy also exhib- was supported by a faculty research grant from Yonsei University College of Medicine (6-2018-0077, 6-2020-0233). its a dual effect on EMT, depending on the setting, either activating or inhibiting it. Through modulating autoph - Data Availability agy signaling pathways, such as integrin, NF-κB, Wnt The data that support the findings of this study are available from the corresponding author upon reasonable request. and TGF-β, the EMT process has a dramatic effect on autophagy modulation [33]. Given the complexity of this Declarations interaction, elucidating the mechanisms of their mutual regulation is challenging, but have clinical benefits in Competing interests cancer treatment. The authors declare no competing interests. This study has limitations that need to be addressed. Ethics approval and consent to participate First, we could obtain only one resistant cell line of NCI- The protocol of this original study was in accordance with the ethical N87R. When we exposed SNU216 and NCI-N87 cells to standards of the responsible committee on human experimentation and with the Helsinki Declaration. After the study was approved by the Institutional trastuzumab, SNU 216 cells were unable to produce resis- Review Board of Gangnam Severance Hospital (IRB 3-2018-0209), clinical tant cells and it was consequently assumed that the SNU samples for organoid establishment and biological analyses were obtained 216 cell line already exhibited trastuzumab-resistance. from patients at Gangnam Severance Hospital with informed consent. In addition, knock-down or knock-out of Wnt3a study is Patient consent for publication required to ascertain whether the Wnt/β-catenin path- Not applicable. way is critical for maintenance of trastuzumab resistance. Our study revealed that trastuzumab-resistant gastric Received: 17 May 2023 / Accepted: 25 September 2023 cancer cells exhibit EMT-like phenotype by promoting the Wnt signaling pathway. 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Journal

BMC CancerSpringer Journals

Published: Sep 29, 2023

Keywords: Wnt; Gastric cancer; Trastuzumab; Resistance; Epithelial to mesenchymal transition

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