Reduced taurine synthesis underlies morphine-promoted lung metastasis of triple-negative breast cancer

doi:10.21203/rs.3.rs-4980929/v1

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Research Article

Reduced taurine synthesis underlies morphine-promoted lung metastasis of triple-negative breast cancer

https://doi.org/10.21203/rs.3.rs-4980929/v1

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Background

The mechanisms underlying the progression and metastasis of triple-negative breast cancer (TNBC) in the context of extended morphine exposure remain poorly understood. Morphine consumption has been a pressing issue in many countries. While the physiological impact of extended morphine use is multifaceted, cancer patients with a history of prolonged morphine usage often result in poor prognosis.

Methods

In this study, we investigated the impact of extended morphine treatment on the transcriptional profiles of TNBC. To this end, mice were administered morphine intraperitoneally for 14 days followed by the implantation of EO771 cells, which are triple negative breast cancer cells, into their mammary fat pad. After primary tumors were removed on 38th day, a subset of mice were continuously giving saline or morphine until the 68th day. Tumor size, organ metastasis, and tumor RNA expression were analyzed.

Results

Our findings showed that prolonged exposure to morphine led to an increase in lung metastasis in the mouse model of triple-negative breast cancer. We conducted RNA sequencing analysis on tumors to compare their transcriptional profiles with or without metastasis. Through pathway analysis, we specifically examined the novel impact of morphine on the downregulation of taurine/hypotaurine biosynthesis. Given that morphine, droperidol (a dopamine receptor antagonist), and naloxone (an opioid receptor antagonist) might act through either opioid receptors or dopamine receptors, we further demonstrated that taurine mitigated EO771 cell invasion induced by morphine, but not by droperidol or naloxone treatment. Additionally, morphine treatment markedly decreased the expression of GAD1, one of the enzymes essential for taurine biosynthesis, whereas droperidol and naloxone did not.

Conclusion

The findings of morphine-induced reduction in GAD1 level and the inhibition of invasion by taurine treatment suggest that taurine could serve as a potential supplement for triple-negative breast cancer patients who require morphine as part of their treatment regimen or due to their circumstances.

cysteine dioxygenase type

glutamate decarboxylase 1

morphine usage

taurine biosynthesis

triple negative breast cancer

As morphine is commonly used for pain treatment, it is also used to treat various diseases, such as coronary artery disease, pulmonary edema and Acquired Immunodeficiency Syndrome [1, 2]. Chronic usage of > 120 mg/day in clinical settings may lead to opioid tolerance or addiction [3]. Breast cancer patients might have been prescribed morphine for an extended duration for reasons such as myofascial pain syndrome or chronic pancreatitis before encountering cancer-related pain. Consequently, they face a considerable risk of tumor progression due to immunosuppression [4, 5].

Morphine binds to µ, κ, and δ opioid receptors, belong to the G-protein-coupled receptor (GPCR) family, and exerts its effects on the peripheral and central nervous systems [6, 7]. The interaction between morphine and GPCRs results in the dissociation of the heterotrimeric G_i/o protein into G_iα/o and G_βγ subunits and subsequent downstream signals. G_iα/o inhibits the activity of protein kinase A, adenylyl cyclase, and the synthesis of cyclic adenosine monophosphate. In turn, the actions of transient receptor potential cation channel subfamily V member 1 and voltage-gated sodium channels are inhibited [8]. G_βγ blocks calcium channels and opens potassium channels. Morphine reduces neural excitability and triggers the release of neural transmitters. extended morphine exposure results in neuronal adaption, such as the decreased expression of mammalian target of rapamycin and the increased expression of Rictor [9, 10].

Breast cancer accounts for 39% of newly diagnosed cancer cases and 44% of deaths in Asian countries and was the third leading cause of death in Taiwan in 2019 [11]. More than 70% of patients with breast cancer experience pain-related symptoms [12]. Those with advanced breast cancer may experience higher pain scores due to metastasis or increased tumor mass. Morphine is an effective drug for treating severe breast cancer–related pain symptom. The morphine dosage needs to be increased as the progression of pain due to tolerance. Recent studies have reported the adverse effect of morphine on tumor growth and metastasis, including in triple-negative breast cancer [13, 14]. Triple-negative breast cancer is characterized by low expression levels of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor and accounts for 10–20% of all breast cancers. The rate of metastasis is higher in triple-negative breast cancer than other breast cancer types, and the survival probability in triple-negative breast cancer is thus lower due to the lack of a therapeutic target. Mechanisms underlying morphine-mediated tumor growth and metastasis remain unclear. Mathew et al. reported that knockout of the µ-opioid receptor (MOR) or the use of a MOR antagonist considerably inhibited tumor growth [15]. Proposed mechanisms include the release of substance P, the up-regulation of survivin expression, and c-Jun N-terminal kinase–mediated activation of the mitochondria-dependent pathway [16–18].

On the other hand, morphine was reported to decrease metastasis and invasion by reducing metalloproteinase activity and weakening endothelial cell adhesion molecule [19, 20]. Nonetheless, clinical data point to a greater potential of morphine on promoting, rather than inhibition, of metastasis [19, 21, 22]. The discrepancy of results may lie in the duration of morphine usage. Most existing research focuses on morphine administered for immediate pain relief, with less attention on the effects of prolonged morphine use or pre-exposure to morphine. For this, studying the impact of extended morphine usage, beyond its conventional role as a painkiller during cancer treatment shall provide unique perspective regarding impact of morphine on cancer progression. The present study aims to compare the gene expression profiles of metastatic tumors induced by extended morphine exposure to those of spontaneous triple-negative breast tumors.

2.1 Reagents

RPMI-1640, fetal bovine serum (FBS), and 6-diamidino-2-phenylindole were purchased from Invitrogen (Carlsbad, CA, USA). Additionally, 3-(4,5 dimethylthiazol-2-tl)-2,3-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (STL, USA). Bovine serum albumin (BSA) was purchased from Santa Cruz Biotechnology (SantaCruz, CA, USA). The SPLInser polyethylene terephthalate membrane (pore size: 8.0 µm) was obtained from SPL Lifesciences (Pocheon, South Korea). Matrigel was purchased from BD Biosciences (San Jose, CA, USA). TRIzol reagent was purchased from Invitrogen. The SYBR green master mix, reverse transcription kit, and StepOnePlus PCR system were purchased from Applied Biosystems (Foster City, CA, USA). Taurine was from Sigma-Aldrich (Saint Louis, MO, USA), Morphine sulfate was from Taiwan Food and Drug Administration, Ministry of Health and Welfare, Taiwan. Droperidol was purchased from the Excelsior Pharmatech Labs (Taipei, Taiwan) and Naloxone was from UniPharma (Taipei, Taiwan).

2.2 Cell culture, animal handling, and ethics statement

EO771 cells [23–25] (obtained from Dr. Tsung-Hsien Chuang, National Health Research Institutes, Taiwan) were maintained in six-well plates with RPMI-1640 supplemented with 10% FBS at 37°C and 10% CO₂. Female, 12–14-week-old C57BL/6 mice were purchased from the National Laboratory Animal Center, Taiwan, and housed in a 12/12-hour light/dark cycle.

Experimental mice (n = 11, C57BL/6Jarl, female) were intraperitoneally injected with morphine (10 mg/kg/day) or equal volume of saline for the indicated days (control group n = 10) (Fig. 1C), and the EO771 cells were implanted into the mammary fat pad (2 × 10⁵ cells in 150 µL of PBS) on the 14th day. Flick tail test was performed 1 hour after 10 mg/kg morphine injection to evaluate delayed nociception to a heat stimulus. Tumor size was measured every 3 days from the 31st day. Thirty-eight days after the first morphine injection, the tumor was resected under general anesthesia (1.5% isoflurane with oxygen). After removing the primary tumors, six of the saline-treated group and four of the morphine-treated group were continuously given saline or morphine and euthanized on the 68th day. Their liver and lung were also harvested to evaluate metastasis on day 68. All animal experiments were conducted in accordance with the guidelines of the Laboratory Animal Center of National Tsing Hua University (NTHU), Taiwan. Animal usage protocols were reviewed and approved by NTHU’s Institutional Animal Care and Use Committee (approval number: 10607).

2.3 Cell proliferation and invasion assays

EO771 cells used for proliferation and invasion assays were the same source and passage numbers. Triplicates were used for each independent experiment. At least three independent experiments were performed. To determine the effect of morphine on cell viability/proliferation, EO771 cells cultured in a 96-well plate were treated with different morphine concentrations (1, 10, or 100 µM) for 48 h. The MTT assays were performed to determine relative cell viability. Morphine stock solution was serial-diluted with RPMI medium (without FBS), thus RPMI was added to the culture as the negative control (0 morphine).

To determine cell invasion, the Boyden chamber assays were performed to analyze cell invasion ability. A transparent polyethylene terephthalate (PET) membrane (8 µm) insert was coated with Matrigel in 24-well plates, EO771 cells were suspended in RPMI (containing 0.1% w/v BSA, 10⁶ cells/mL) and plated onto solidified Matrigel. RPMI medium (supplemented with 10% FBS) was added to the lower chamber as a chemoattractant to attract cell invasion. After incubation for 26 h, cells migrated to the bottom side of PET membrane were fixed with 4% paraformaldehyde and stained with crystal violet. Images of crystal violet-stained cells were taken using Zeiss Observer Z1 microscope (Zeiss, Jena, Germany). Number of cells was counted using Image J software (https://imagej.nih.gov/ij/index.html). Relative invasion ability was calculated as cell number per chamber and normalized to “0 morphine” control (= 1).

2.4 Real-time quantitative PCR

RNAs of EO771 cells were collected using TRIzol and mRNAs (2 µg) were reverse transcribed to cDNA. Real-time PCR with SYBR Green detection was performed using an ABI PRISM 7500 sequence detection system (Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control. Primers were listed below: GAD1_F: 5’-GCG GGA GCG GAT CCT AAT A-3’; GAD1_R: 5’-TGG TGC ATC CAT GGG CTA C-3’; CDO1_F: 5’-GAG GGA AAA CCA GTG TGC CTA C-3’; CDO1_R: 5’-CCT GTT CTC TGG TCA AAG GCG T-3’; GAPDH_F: 5’-ATG TTT GTG ATG GGT GTG AA-3’; GAPDH_R: 5’-ATG CCA AAG TTG TCA TGG AT-3’.

2.5 RNA-sequencing and data analysis

Total RNAs were extracted from collected tumors with a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Agilent 2100 Bioanalyzer–with Agilent RNA 6000 Nano kit was used to determine the quality of RNAs followed by the RNA sequencing using Illumina HiSeq 4000, conducted by Genomics, Taiwan (https://www.genomics.com.tw/). Purified RNA was used for preparation of the sequencing library by using the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA) in accordance with the manufacturer’s instructions. Briefly, mRNA was purified from total RNA (1 µg) by using oligo (dT)-coupled magnetic beads and fragmented into small pieces under elevated temperature. First-strand cDNA was synthesized using reverse transcriptase and random primers. After the generation of double-stranded cDNA and adenylation on the 3′-end of DNA fragments, adaptors were ligated and purified using the AMPure XP system (Beckman Coulter, Beverly, USA). The quality of the libraries was examined using the Agilent Bioanalyzer 2100 system and a real-time polymerase chain reaction system. The qualified libraries were sequenced on an Illumina NovaSeq 6000 platform with 150-bp paired-end reads generated by Genomics (BioSci & Tech Co., New Taipei City, Taiwan). All experiments were performed in Genomics Core through the Genomics Core laboratory process (https://en.genomics.com.tw/).

The low-quality bases and sequences from adapters in raw data were removed using Trimmomatic (version 0.39) [26]. The filtered reads were aligned to reference genomes by using Bowtie2 (version 2.3.4.1) [27]. The user-friendly software RSEM (version 1.2.28) was used for quantification of transcript abundance [28]. Normalization and identification of differentially expressed genes (DEGs) were carried out by using the R package edgeR. we applied generalize linear model to estimated effect sizes and p-values of differentially expressed genes between morphine and saline treatments [29, 30]. The functional enrichment analysis of Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways among gene clusters was performed using an R package called ClusterProfiler (version 3.6.0) [31–33] and analyzed using an online bioinformatics tool (Database for Annotation, Visualization, and Integrated Discovery, DAVID; https://david.ncifcrf.gov) [34]. The schematic diagram of data analysis is illustrated in Fig. 2.

To determine the relationship between the selected hub genes or pathways and survival, we compared the identified hub genes and pathways by using data from The Cancer Genome Atlas (TCGA) database. The UCSC Xena website (https://xenabrowser.net) is an online database used for determining the correlations among genotypes, phenotypes, and survival in TCGA breast cancer dataset. We applied hub and transcription genes in UCSC Xena to determine the association of overall survival with gene expression. Patients were divided into high and low gene expression groups for the survival analysis. For the pathway analysis, we determined the total number of genes involved in each pathway and analyzed the relationship between relapse-free survival and pathway expression [35].

2.6 Statistical Analysis

Difference between un-treated and different morphine dose-treated samples for MTT and invasion assays was determined by one sample Student’s t-test. For multiple comparison, we adjusted P value by bonfernonni method. Each morphine-treated sample was normalized to untreated sample (= 1) (Fig. 1A-B).

We evaluated the difference of tumor volumes between saline- and morphine-treated groups for 17, 20, 23 days. In order to conform to a normal distribution, we excluded outliers which were outside mean±1.5 standard deviation, and used Student’s t-test and two-way ANOVA (Fig. 1D). These statistical methods were used by R (version 4.0.2).

Relative in vitro invasion analysis was performed using Boyden chamber assays and used Student’s t-test to determine P value (Figs. 1, 6).

3.1 Effect of morphine on cell viability, invasion, tumor growth, and metastasis

Triple-negative breast cancer is highly metastatic and has poorer prognosis than other breast cancer subtypes [36]. The EO771 cell line is derived from the C57 BL/6 mouse model of spontaneous breast cancer, and this is an established model for studying triple-negative breast cancer [37]. To investigate the effect of extended morphine usage on breast cancer cells, EO771 cells were incubated with morphine at the concentrations of 0, 1, 10, or 100 µM for 48 h. Our results revealed that up to 100 µM morphine did not affect cell proliferation compared with the mock-treated control. The Boyden chamber assays were performed to examine the effect of morphine on the invasion ability of the EO771 cells within 26 h. The EO771 cells treated with ≥ 1 µM morphine exhibited at least twice the invasion ability of the control group (Fig. 1A-B). These results suggest that morphine significantly enhances the invasion ability of EO771 cells but does not affect their proliferation.

While morphine is generally used for pain management, this study focuses on the effect of extended use of morphine on cancer progression, thus no additional pain-initiating agent or pain-relief medicine were used to avoid drug-drug interaction. To mimic the effects of extended morphine usage in humans, the mice were intraperitoneally administered 10 mg/kg morphine or same volume of saline daily for 2 weeks before EO771 cell implantation to fat pad. The tail-flick test was used to ensure delayed nociception to a heat stimulus for morphine-treated mice. Figure 1C presents a schematic diagram of the experimental approach including morphine injection and tumor measurement. Morphine pre-treatment resulted in a significant increase of the tumor volume. Significant differences in tumor volumes were observed between the saline-treated group and the morphine-treated group on days 17, 20, and 23 after orthotopic injection of EO771 cells. These findings suggest that morphine administration may have a measurable impact on tumor growth dynamics (Fig. 1D). After removing primary tumors on day 38, 6 saline-treated mice and 4 morphine-treated mice were followed-up, based on the health condition of mice, to monitor potential metastasis till day 68. On the 54th day after tumor cell implantation, lung metastasis developed in 3 out of the 6 mice in the saline group and in all 4 mice in the morphine-treated group (Fig. 1E). The incidence of lung metastasis was higher in the morphine-treated group compared to saline-treated group. The averaged total area of tumors in lung, on the other hand, was increased in morphine-treated mice, but without significant difference (Fig. 1F). The total area of tumor growth in lung represents colonization of metastasized tumors. Thus, these results suggest that morphine promotes metastasis process but not colonization.

To determine the mechanism through which morphine affects tumor formation and metastasis, total RNA were isolated from tumors for RNA sequencing analysis. DEGs were compared between the morphine-treated and control groups. RNA samples extracted from primary tumors were designated the control saline 1 (S1), and tumors from spontaneous lung metastasis were designated the saline 2 (S2) groups. Tumor RNA samples extracted from morphine-treated mice at day 38 were morphine 1 (M1). A subset of mice were followed-up for metastasis and RNA were isolated from tumors metastasized to lung, designated morphine 2 (M2).

3.2 Differential expression genes, functional and pathway enrichment analysis

Different from the previous reports on morphine-mediated tumor growth[38, 39], the current study focuses on the effects of extended morphine treatment on TNBC metastasis. We focused on the comparison of differential expression genes between M2 and S1 (M2/S1 and M2/S2) based on the whole genome RNA-seq data. The volcano plots illustrated the distribution of up-regulated and down-regulated genes (Fig. 3). In the M2/S1 analysis, differentially expressed genes (DEGs) were identified, among which 401 genes were recognized as dominant genes (false discovery rate [FDR] < 0.05). Of these 401 genes, 32 hub genes were up-regulated (Log₂ FC > 1) and 225 hub genes were down-regulated (Log₂ FC < − 1) (Fig. 3A). In addition, 12,586 genes were differentially regulated when comparing metastatic M2/S2 tumors. There were 93 dominant up-regulation hub genes (FDR < 0.05, Log₂ FC > 1) and 63 down-regulation hub genes (FDR < 0.05, Log₂ FC < 1), and volcano plot showed a relatively symmetrical distribution of up-regulated and down-regulated genes (Fig. 3B).

We used the DAVID tool to perform the DEG enrichment analysis, including GO term and KEGG analyses. For RNA seq analysis of M2/S1, we determined the GO terms and KEGG pathways of the identified 257 genes by using DAVID. In the GO analysis, enrichment processes were divided into biological process (BP), molecular function (MF), and cellular compartment (CC) categories for up-regulated genes and down-regulated genes. (Tables S1 and S2). One up-regulation and 10 down-regulation pathways of KEGG were listed (Table S3). In the M2/S2 group, KEGG analysis revealed 2 up-regulation pathways of KEGG, and no pathways were identified as significantly down-regulated (Table S4). The expression of genes involved in regulation of the extracellular matrix was observed to be decreased in this study, consistent with common findings for cancer progression. Genes involved in lipid metabolism, protein/peptide metabolism, and signal transduction in the KEGG analysis were compatible with those identified through GO analysis.

Based on the M2/S1 down-regulated genes, 225 genes were selected for the subsequent analysis (FDR < 0.05). The results of the DAVID analysis revealed 10 down-regulated pathways after extended morphine treatment and the relative expression of genes (S1, S2, and M2 counts) within each pathway were shown (Fig. 4A). We then used the Cytoscape plugin ClueGo to integrate the functional pathways identified in the GO term and KEGG analyses of M2/S1 group. Four dominant KEGG pathways and top 20 GO terms were illustrated in Fig. 4B. The GO term analysis indicated the dominant down-regulation of pathways by morphine treatment. The KEGG analysis revealed dominant down-regulation of arachidonic acid, lipolysis, the renin–angiotensin system, and taurine/hypotaurine metabolism. The variation in KEGG pathways does not elucidate the mechanisms underlying tumor metastasis. In comparing the dominant hub genes of the M2/S1 and M2/S2 groups, 16 hub genes were uniformly up-regulated, and 29 were uniformly down-regulated. Of the 45 hub genes analyzed, only four were exclusive to either the M2/S1 or M2/S2 KEGG pathways. One hub gene (Hspa1b) associated with upregulation and three with downregulation (Gad1, Mrvi1, and Cd1d1) were involved in the M2/S1 KEGG pathways, while no identical hub genes were found in the M2/S2 pathways (Table S5). Hspa1b, which encodes a heat-shock protein, is involved in the up-regulated KEGG pathway associated with Legionellosis. Up-regulation of this pathway has been reported as a factor for poor outcomes [40]. GAD1 encodes the key enzyme for taurine synthesis and participates in the KEGG pathway of taurine/hypotaurine metabolism. The Mrvi1 protein is implicated in the cGMP-PKG signaling pathway and may relate to poor survival[41]. CD1d1 and CD1d2 co-encode the CD1d protein, which is critical for the development of natural killer T cells, and down-regulation of CD1d would promote breast cancer metastasis[42, 43]. Notably, one novel discovery is that morphine influences GAD1 expression; GAD1 is a crucial protein in taurine/hypotaurine metabolism and is associated with the three enzymes responsible for taurine biosynthesis.

The findings of ClueGo analysis revealed that down-regulated KEGG pathways—arachidonic acid metabolism, regulation of lipolysis in adipocyte, renin–angiotensin system, and taurine and hypotaurine metabolism—correlated with a decreased survival rate (Fig. 4). The findings revealed an involvement of taurine and hypotaurine metabolic processes in morphine regulation. To determine the role of taurine synthesis genes in breast cancer patients, we analyzed the candidate genes by using TCGA Breast Cancer (BRCA) datasets (n = 1097). The BRCA dataset is a cohort database and includes information on variations in gene copy number (n = 1080), DNA methylation (n = 345), and phenotypes (n = 1236). In the TCGA database, higher gene expression levels associated with taurine and hypotaurine metabolism correlated with improved survival outcomes (Fig. 5). Based on these findings, we hypothesized that taurine deficiency may contribute to TNBC progression and increased lung metastasis.

3.3 Effect of taurine on morphine-mediated cell invasion

If the effect of reduced taurine and increased invasion are morphine-opioid receptor-specific action, an antagonist of opioid receptor, naloxone, is not expected to exert similar effect. Moreover, morphine was previously reported to trigger physiological action through dopamine receptor[44]. If this is true, a dopamine receptor antagonist, droperidol, would reduce invasion ability. To this end, EO771 cells were treated with morphine (M), morphine + taurine (M + T), naloxone (N), naloxone + taurine (N + T), droperidol (D), droperidol + taurine (D + T), and taurine alone (T) over a 4-day period to assess their individual and combined impacts on cellular invasion. As shown in Fig. 6A, there was a significant increase of cell invasion with morphine treatment compared to controls. This effect was mitigated when taurine was added (M + T), highlighting the potential of taurine to counteract morphine-induced cellular invasion. No significant differences were observed among the D, N, untreated, and T groups, nor between the treatments with and without taurine (D vs. D + T and N vs. N + T). Treatment of N and D did not significantly increase invasion either. Extended treatment of morphine similarly increased cell invasion (Fig. S1). Nonetheless, longer in vitro treatment inevitably involved multiple cell trypsinization and re-plating, confounding effect of cell cycle may have a role on cell behavior. These results reveal a distinct mechanism by which morphine-opioid receptor-specific action influences pro-invasive behavior of cancer cell that is reversed by taurine.

Based on our RNA-sequencing results, one key enzyme of taurine biosynthesis, GAD1, was significantly reduced in tumors derived from morphine-treated mice with lung metastasis (M2) compared to those from saline-treated mice with lung metastasis (S2). Another enzyme, cysteine dioxygenase 1 (CDO1), was not different between these two groups (Fig. 6B). To determine whether morphine treatment modulates the expression of these enzymes, EO771 cells treated with morphine, droperidol, or naloxone for 4 days, only morphine treatment reduced expression of GAD1 by 20%. Consistent with our RNA-sequencing results, CDO1 expression was not affected by morphine, droperidol, or naloxone (Fig. 6C). Compared to the in vivo tumor data, morphine treatment showed more profound reduction (50%) of GAD1 (Fig. 6B-C).

Together, these analysis highlight the novel role of extended morphine treatment in modulating the invasive behavior of TNBC cells through GAD1 gene expression and demonstrate the potential of taurine as a therapeutic adjuvant to mitigate the invasive effects associated with morphine treatment.

Whether morphine promotes or inhibits tumor formation remains controversial [45], possibly due to diversity of tumor types, stages and durations of morphine usage. As morphine has been implicated in immune modulation [46, 47], this role in cancer metastasis is not clear. Findings from this study showed a prolonged morphine treatment increased lung metastasis of TNBC which correlated to reduced expression of GAD1, encoding the key enzyme for taurine biosynthesis. Taurine is an abundant free amino acid and is involved in anti-inflammation and tumor suppression [48]. In addition, taurine was reported to enhance the efficacy of chemotherapy and induce cancer cell apoptosis [49]. The mechanism of anti-tumor effect of taurine may be through suppressing extracellular-signal-regulated kinase/ribosomal S6 kinase signaling [48–50]. Thus, decreased taurine level is likely associated with poor prognosis. A very recent report showed that diminished taurine level can lead to exhaustion of CD8⁺ T cells, whereas supplementation with taurine may enhance the efficacy of therapy[51]. In line with our study, we showed that extended treatment of morphine increased lung metastasis for TNBC. Silencing GAD1 genes was reported as the poor prognosis of patients with renal cell carcinoma [52]. Consistent with these findings, our results indicated that extended morphine usage led to decreased GAD1 expression in TNBC, which maybe the key mechanism underlying the reduction in taurine level and thus the promotion of lung metastasis (Fig. 7). Through human triple-negative breast cancer dataset analysis, GAD1 expression is stage-dependent, with an initially increase and then decreased at stage IV comparing metastatic versus non-metastatic cases (Fig. S2). Our report reveals an extended exposure to morphine for TNBC patients is a risk factor. Extended morphine exposure appears to affect GAD1 expression and reduce taurine specifically within the cancer tissue microenvironment, rather than exerting systemic effects. This localized reduction complicates the assessment of taurine variability in TNBC patients.

This study reveals one of the mechanisms that underlies morphine-mediated metastasis of triple negative breast cancer through reducing the level of taurine biosynthesis enzyme, GAD1. Based on our findings, we recommend taurine supplementation for long-term morphine exposure of TNBC patients.

GPCR G-protein-coupled receptor

MOR m-opioid receptor

NK natural killer

FBS fetal bovine serum

MTT 3-(4,5 dimethylthiazol-2-tl)-2,3-diphenyltetrazolium bromide

BSA Bovine serum albumin

NTHU National Tsing Hua University

GAPDH Glyceraldehyde-3-phosphate dehydrogenase

FDR False Discovery Rate

KEGG Kyoto Encyclopedia of Genes and Genomes

GO Gene Ontology

DEGs Differentially expressed genes

DAVID Database for Annotation, Visualization, and Integrated Discovery

TCGA Cancer Genome Atlas

GAD1 Glutamate decarboxylase 1

BP Biological process

MF Molecular function

CC Cellular compartment

CDO1 Cysteine dioxygenase 1

CD1d1 Cluster of Differentiation 1-d1

Hspa1b Heat Shock Protein a1b

Mrvi1 Murine Retrovirus Integration site 1

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work is supported by the Ministry of Science and Technology, Taiwan (grant#: 108-2320-B-007-005-MY3 and 111-2311-B-007-012 to Dr. Linyi Chen) and National Tsing Hua University, Taiwan (grant#: 110F7MBNLE1 to Dr. Shih-Hong Chen). This manuscript was edited by Wallace Academic Editing

Availability of data

The datasets generated and/or analyzed during the current study are available in the Figshare repository, DOI 10.6084/m9.figshare.22644256

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Funding

This work was supported by the National Health Research Institutes, Taiwan under Grant#: NHRI-EX110-10813NI) the Ministry of Science and Technology, Taiwan, under Grant#: MOST 108-2320-B-007-005-MY3; 111-2311-B-007-012 to Linyi Chen and by National Tsing Hua University, Taiwan, under Grant#:110F7MAFE1 to Linyi Chen and Shih-Hong Chen.

All authors didn’t use AI assisted-technology.

Ethics approval and consent to participate

Animal usage protocols were reviewed and approved by NTHU’s Institutional Animal Care and Use Committee (approval number: 10607).

Author contribution and contributor statement

Shih-Hong Chen: Data analysis, Investigation, Visualization, Writing – original draft, review & editing, Funding. Chien-Hung Shih: Animal study, quantification of tumor area in lung tissue, Writing –original draft. Ting-Ling Ke: Conducted all experiments for revision. Data analysis, Writing – review and editing. Zi-Xuan Huang: Tumor RNA preparation. Kuo-Chin Chen: TCGA datasets analysis, Software, Visualization. Chia-Ni Hsiung: Software, Visualization, Statistical analysis. Tsung-Hsien Chuang: Cell line, Methodology. Li-Kuei Chen: Conceptualization, Provide reagents, Funding. Linyi Chen: Conceptualization, Investigation, Visualization, Writing –original draft, review & editing, Funding.

Linyi Chen is responsible for the overall content as guarantor, accepts full responsibility for the finished work, the conduct of the study, had access to the data, and controlled the decision to publish.

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No competing interests reported.

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Reduced taurine synthesis underlies morphine-promoted lung metastasis of triple-negative breast cancer

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Abstract

Figures

1. Background

2. Methods and Materials

2.1 Reagents

2.2 Cell culture, animal handling, and ethics statement

2.3 Cell proliferation and invasion assays

2.4 Real-time quantitative PCR

2.5 RNA-sequencing and data analysis

2.6 Statistical Analysis

3. Results

3.1 Effect of morphine on cell viability, invasion, tumor growth, and metastasis

3.2 Differential expression genes, functional and pathway enrichment analysis

3.3 Effect of taurine on morphine-mediated cell invasion

4. Discussion

5. Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1