Effect of Wnt5a on drug resistance in estrogen receptor-positive breast cancer

Background Previously, we reported that Wnt5a-positive breast cancer can be classified as estrogen receptor (ER)-positive breast cancer; its prognosis is worse than that of Wnt5a-negative breast cancer. This study aimed to investigate the mechanisms underlying the poor prognosis in Wnt5a-positive breast cancer patients. Methods In total, 151 consecutive ER-positive breast cancer patients who underwent resection between January 2011 and February 2014 were enrolled. DNA microarray and pathway analyses were conducted using MCF-7 cells stably expressing Wnt5a [MCF-7/Wnt5a (+)]. Based on the outcomes, cell viability/drug sensitivity assays, and mutation analysis were performed using cell cultures and breast cancer tissues. The relationship between Wnt5a and the PI3K–AKT–mTOR signaling pathway was also examined. Results The relapse-free survival rate in patients with Wnt5a-positive breast cancer was significantly lower than that in patients with Wnt5a-negative breast cancer (P = 0.047). DNA microarray data suggest that only the cytochrome P450 (CYP) pathway was significantly upregulated in MCF-7/Wnt5a (+) cells (P = 0.0440). Additionally, MCF-7/Wnt5a (+) cells displayed reduced sensitivity to the metabolic substrates of CYP, tamoxifen (P < 0.001), paclitaxel (P < 0.001), and cyclophosphamide (P < 0.001). Of note, PIK3CA mutations were not associated with the expression of Wnt5a in breast cancer tissue and culture cells. Conclusions In ER-positive breast cancer, Wnt5a upregulates the CYP metabolic pathway and suppresses tamoxifen, paclitaxel, and cyclophosphamide resistance, all of the three, standard treatment methods for ER-positive breast cancer. Wnt5a is thus potentially involved in the poor prognosis of ER-positive breast cancer independently of the PI3K–AKT–mTOR signaling pathway. Supplementary Information The online version contains supplementary material available at 10.1007/s12282-021-01241-0.

Ovarian cancer cells overexpressing Wnt5a showed low chemosensitivity to paclitaxel and epirubicin [9]. However, in breast cancers, relatively few studies have examined the association between Wnt5a and drug sensitivity. Furthermore, upregulated PI3K-AKT-mTOR signaling is associated with a poor prognosis in ER-positive breast cancer [10][11][12]. Of note, Wnt5a is reportedly upregulated in ER-positive breast cancers harboring a PIK3CA mutation [13], indicating a close relationship between PI3K signaling and the Wnt5a pathway. Therefore, in the present study, the signaling pathways associated with Wnt5a were investigated to analyze the mechanisms underlying a Wnt5a-mediated drug resistance and poor prognosis.

Patients and breast cancer tissues (RFS-tracked cases)
Consecutive breast cancer tissues resected between 2011 and 2014 were reviewed as previously described [8]. The followup period was extended, and the 8-year RFS was investigated by December 2019 in the present study. To strictly investigate "recurrence of early-stage breast cancer," the two patients with Stage IV at diagnosis included in our previous study [8], were excluded from this study.
Furthermore, we grouped patients treated with the key drugs, tamoxifen, paclitaxel, cyclophosphamide, epirubicin and 5-fluorouracil, and investigated RFS comparing Wnt5apositive and -negative patients.

Western blot analysis
For immunoblot analysis, MCF-7 and MDA-MB-175-VII cells were washed with PBS and lysed with lysis buffer containing a Phosphatase Inhibitor Cocktail (Nacalai Tesque Inc., Kyoto, Japan). Proteins were separated via SDS-PAGE and then electro-transferred onto nitrocellulose membranes (Amersham Protran Premium, GE Healthcare, Buckinghamshire, UK). The membranes were probed with various primary and secondary antibodies (Online Resource 1B) and visualized with enhanced chemiluminescence detection reagents (Amersham ECL Select, GE Healthcare, Buckinghamshire, UK). All western blotting experiments were performed in triplicate.

Detection of PIK3CA mutant variants
Among the 151 cases immunoreactive for Wnt5a, PIK3CA mutations were evaluated only in those with a tumor size of ≥ 1 cm in diameter. The QIAamp DNA FFPE Tissue Kit (Qiagen GmbH, Hilden, Germany) was used to extract DNA from formalin-fixed paraffin-embedded (FFPE) tissues. The E542K, E545D/K, and H1047R/L were detected through direct sequencing using the primers listed in Online Resource 1C.

Quantification of Wnt5a mRNA expression
RNA was extracted using the NucleoSpin total RNA FFPE (Takara Bio, Shiga, Japan) from tissues sections sliced from the FFPE block, including the tumor component only. cDNA was synthesized through reverse-transcription using the Pri-meScript II High Fidelity RT-PCR Kit (Takara Bio). Wnt5a expression was quantitatively analyzed via real-time PCR using the SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA, USA) and the CFX96 real-time PCR detecting system (Bio-Rad). Wnt5a expression was quantified using the ΔCt value. The used primers are listed in Online Resource 1C.

Statistical analysis
Statistical analysis was performed using the EZR [14] and SPSS (Version 20.0, Chicago, IL, USA) software. Welch's t test was used to compare the age, and cell viability between Wnt5a-negative and -positive cells, and Wnt5a-silenced and -non-silenced cells. The clinicopathologic characteristics were analyzed using the Chi-squared test. The significance between RFS curves was analyzed using the generalized Wilcoxon test. The frequency of Wnt5a positivity and the expression levels of Wnt5a mRNA were compared between PIK3CA mutation-negative and -positive cases using the Chi-square test and Welch's t-test, respectively. P values < 0.05 were considered statistically significant.

The CYP pathway is upregulated in Wnt5a-expressing MCF-7 cells
To investigate pathways related to the recurrence of Wnt5apositive breast cancer, MCF-7 cells stably expressing Wnt5a were established and DNA microarray analyses were performed. A total of 176 candidate genes differentially expressed between MCF-7/Wnt5a (+) and MCF-7/Wnt5a (−) cells, were identified; 76 unique genes were upregulated (expression ≥ twofold) and 100 unique genes were downregulated (expression ≤ 1/twofold; Online Resource 4). The P-value was determined using the DAVID database (Table 1). Interestingly, as per the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, physiological pathways directly or indirectly influencing Wnt5a expression were identified; only the cytochrome P450 (CYP) pathway, involved in drug metabolism [15], was identified among the upregulated genes (P = 0.0440; Table 1). Conversely, certain virus-related pathways were identified among the downregulated genes (Table 1). Interestingly, the JAK-STAT signaling pathway, associated with cancer progression, was attenuated in MCF-7/Wnt5a (+) cells (Table 1). Of note, the upregulated genes were primarily involved in the oxidation-reduction process, response to calcium ions, retinoid metabolic process, response to

Relationships between differentially expressed genes, cancer, and CYP
The 176 differentially expressed genes are represented in the form of a heatmap (Fig. 2). Among them, 21 genes are related to breast cancer/cancer/CYP and have been reported to promote cell migration and proliferation, and cancer metastasis. The upregulated genes that correlate positively with the malignancy of ER-positive breast cancer are UGT2B15 [16], AKR1C3 [17], ALCAM [8], and PTPRN2 [18]; the downregulated genes are JAK2, STAT1 [19], CCNA1 [20]. Of note, UGT2B15 and AKR1C3 are genes related to the CYP pathway, and ALCAM and PTPRN2 are genes co-expressed with CCNA1 (Online Resource 5).

Wnt5a expression decreases the sensitivity to tamoxifen, paclitaxel, and cyclophosphamide
Tamoxifen, paclitaxel and cyclophosphamide, but not epirubicin and 5-fluorouracil, are metabolized by CYP [21][22][23][24][25]. Therefore, the sensitivity to these five drugs, used in the standard treatment of ER-positive breast cancer, was examined. MCF-7/Wnt5a (+) cells exhibited significantly lower sensitivity to tamoxifen, paclitaxel, and cyclophosphamide than MCF-7/Wnt5a (−) cells at both 48 h and 72 h (P < 0.001; Fig. 3a-c). In contrast, no difference was found in resistance to epirubicin (P = 0.12) and MCF-7/ Wnt5a (+) cells exhibited higher sensitivity to 5-fluorouracil (P = 0.021; Fig. 3d, e). Importantly, similar trends were observed in the context of MDA-MB-175-VII cells, although without significance in some instances (Online Resource 6). Additionally, we examined the difference in the RFS between Wnt5a-positive versus -negative breast cancer patients treated with the different above drugs. The RFS probability was lower in the Wnt5a-positive. However, used. However, no significant differences were detected, probably due to the small sample-size (Online Resource 7).

The PI3K signaling pathway is not correlated with Wnt5a expression
Next, the relationship between the expression of Wnt5a and the PI3K and JNK signaling pathways was examined via western blotting in MCF-7/Wnt5a (+) and MCF-7/Wnt5a (−) cells. The expression of phosphorylated JNK, which occurs downstream of the Wnt5a signaling pathway [2], remained unaltered in Wnt5a overexpressing or silenced cells (Fig. 4a). Similarly, there was no difference in the expression of phosphorylated AKT (Fig. 4b).
PIK3CA mutations were examined in 40 cases (Table 2) and detected in 19 cases of ER-positive breast cancers (Table 3); three principal mutation sites were identified: E542K, E545K, and H1047R [26] (Fig. 5a). Of note, PIK3CA mutations were observed in 8 and 11 Wnt5a-positive and -negative breast cancer patients, respectively. However, there was no significant difference in the frequency of PIK3CA mutations depending on the expression of Wnt5a (P = 0.73; Table 3). Furthermore, no difference in Wnt5a expression was observed depending on the mutation site (Table 4).

Discussion
The recurrence rate of Wnt5a-positive breast cancer patients is significantly higher than that of Wnt5a-negative breast cancer patients. Therefore, this study investigated the association between the expression of Wnt5a expression and malignancy grade and prognosis. Interestingly, pathway analysis revealed that the CYP metabolic pathway was upregulated after Wnt5a overexpression. CYP is a key enzyme that oxidizes various substrates and primarily metabolizes drugs in the liver. In our study, CYP upregulation reduced the sensitivity to tamoxifen, paclitaxel, and cyclophosphamide (all metabolized by CYP). Conversely, the sensitivity to epirubicin and 5-fluorouracil (not metabolized by CYP) was not affected. These results suggest that Wnt5a enhances the tamoxifen, paclitaxel, and cyclophosphamide metabolism via CYP, thus decreasing their intracellular concentration. Therefore, the early termination of adjuvant drug therapy for breast cancer may lead to a high recurrence rate of Wnt5a-positive breast cancer. Importantly, as no prior study have shown the association between Wnt5a and CYP in any cancer, these results are novel.
The genes associated in this study with Wnt5a in ERpositive breast cancer are summarized in Fig. 6 together with the previously reported genes [27][28][29][30]. Among the upregulated genes, UGT2B15 and AKR1C3 are included in the CYP metabolic pathway as per KEGG pathway analyses, suggesting the association between the CYP and Wnt5a pathways in the context of drug sensitivity [16,17]. Interestingly, ALCAM and PTPRN2 were also shown to correlate with Wnt5a expression and to promote cell migration [8,18].   Additionally, the downregulation of the JAK-STAT signaling pathway was reported to contribute to breast cancer progression and metastasis [19]. Of note, it has been reported that the downregulation of CCNA1 leads to cell growth in the context of breast cancer [20]. Interestingly, here we determined that CCNA1 is co-expressed with ALCAM, PTPRN2, and AKR1C3, suggesting an association between them.
Based on our previous reports [12,31,32] we hypothesized that Wnt5a expression would be associated with the PI3K-AKT-mTOR signaling pathway. However, unexpectedly, no significant correlation was found. These results indicate the involvement of pathways other than PI3K in the recurrence of Wnt5a-positive breast cancer.
Altogether, our results indicate that Wnt5a potentially serves as a drug resistance marker in ER-positive breast cancer. However, further studies, in vivo, are essential to validate our results. Additionally, further studies are required for the development of new drugs targeting drug resistance markers; for instance, the development of a treatment strategy for Wnt5a-positive breast cancer based on anti-Wnt5a antibodies, in line with that developed for Wnt5a-positive gastric cancer [33] should be carried out.
In conclusion, here, we show that Wnt5a upregulates CYP expression and diminishes the sensitivity to key drugs used for treating ER-positive breast cancer, including tamoxifen, paclitaxel, and cyclophosphamide. Wnt5a is potentially involved in the poor prognosis of ER-positive breast cancer independently of the PI3K-AKT-mTOR signaling pathway.
Author contributions All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by AA, TK, and MI. The first draft of the manuscript was written by AA and all authors commented on the previous versions of the manuscript. All authors read and approved the final version of the manuscript.

Conflict of interest
The authors have no competing interests to declare.

Ethical approval
The study was approved by the institutional review board of Hiroshima University Hospital (No. 926) and was performed in accordance with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
Informed consent Informed consent was obtained from all participants included in the study.
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:// creat iveco mmons. org/ licen ses/ by/4. 0/. Fig. 6 The genes associated with Wnt5a in ER-positive breast cancer are summarized. The solid black lines represent the relationships already reported in the literature; the colored solid lines and the dotted lines and yellow line represent the relationships defined in this study. TAM tamoxifen, PTX paclitaxel, CPA cyclophosphamide