N6-methyladenosine modification of DYNLT1 Facilitates Tamoxifen Resistance in Luminal B Breast Cancer

doi:10.21203/rs.3.rs-5295457/v1

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Research Article

N6-methyladenosine modification of DYNLT1 Facilitates Tamoxifen Resistance in Luminal B Breast Cancer

https://doi.org/10.21203/rs.3.rs-5295457/v1

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Purpose

Resistance to tamoxifen poses a significant clinical challenge in the management of Luminal B breast cancer, necessitating the identification of novel biomarkers for predicting treatment response and prognosis. The specific role of DYNLT1 in endocrine response within Luminal B breast cancer remains uncertain.

Methods

The expression levels of DYNLT1 were assessed in breast cancer samples using immunohistochemistry, real-time PCR, and western blot analysis. The potential role of DYNLT1 in promoting resistance to tamoxifen was investigated through cell viability and colony formation assays. Furthermore, an in vivo mammary fat pad model was employed to examine the impact of DYNLT1 on tamoxifen resistance in breast tumors. Additionally, luciferase activity assays were conducted to explore the activation of the ER signaling pathway. The enrichment of ELAVL1 on mRNA of DYNLT1 was detected utilizing RNA immunoprecipitation assay.

Results

This study demonstrated that the DYNLT1 expression was particularly upregulated in the Luminal B subtype of breast cancer tissues. Notably, elevated DYNLT1 expression was associated with poorer relapse-free survival among Luminal B breast cancer patients treated with tamoxifen. Functionally, increased DYNLT1 expression induced resistance to tamoxifen both in vivo and in vitro. Additionally, upregulation of DYNLT1 significantly promoted ligand-independent activation of the ER signaling pathway. ELAVL1-mediated m6A modification led to overexpression of DYNLT1 and facilitated the acquisition of tamoxifen resistance phenotype.

Conclusion

Overall, these findings highlight that DYNLT1 could potentially act as a novel biological marker for predicting the effectiveness of tamoxifen treatment and patient prognosis in Luminal B breast cancer.

DYNLT1

Luminal B breast cancer

Tamoxifen resistance

ELAVL1

m6A modification

Breast cancer is molecularly categorized by different subsets of genetic and epigenetic abnormalities into 4 primary subtypes: Luminal A, Luminal B, HER2-enriched, and Basal-like breast cancers [32]. About 70% of breast tumors are of the luminal subtype, which has the most favorable prognosis and responds effectively to endocrine therapies [40]. Further categorization of the luminal subtype into less aggressive Luminal A and more aggressive Luminal B subtypes may be performed via the use of gene expression profiling and the proliferation marker protein Ki-67 [28]. Research has shown that the characteristics of Luminal B cancers differed from those of Luminal A tumors, including younger age of diagnosis, vascular invasion, involvement of positive lymph nodes, larger tumors, and higher grade [4]. In multivariate analysis for relapse-free survival (RFS), Luminal B had hazard ratios that were considerably greater than those of Luminal A [16]. Additionally, Luminal B tumors frequently exhibit decreased or absent progesterone receptor (PR) expression, reduced levels of estrogen receptor (ER) or estrogen-regulated genes, upregulated levels of proliferation-associated genes, and activation of growth factor receptor signaling pathways, which include PI3K/AKT/mTOR and IGF-1R [6, 34]. Importantly, in comparison to Luminal A tumors, the Luminal B subtype appears to be more susceptible to chemotherapy and less responsive to endocrine therapy [1, 11, 36]. Although there are several means to deal with Luminal B breast cancer, many patients develop endocrine resistance as the disease progresses.

N-6-methyladenosine (m6A) is the most abundant epitranscriptomic modification of RNAs, including mRNA and ncRNA, in eukaryotic cells [15]. m6A modification is known to be a reversible dynamical process affecting different dimensions of RNA expression, including regulation of RNA stability, splicing, translation efficiency, and degradation [7]. Dysregulation of m6A-based RNA modification has been responsible for the various stages of carcinogenesis including onset, metastatic spread, relapse, and drug resistance [13, 49]. For example, in MCF7 breast cancer cells, upregulation of the m6A reader HNRNPA2B1 led to an increase in endocrine resistance, cell motility, and stem cell characteristics. In contrast, tamoxifen sensitivity was reestablished in LY2 and LCC9 cells when HNRNPA2B1 was knocked down [35]. Luminal B subtype patients with m6A regulators were more likely to experience disease recurrence, which is consistent with an increased concentration of immune-associated genes and cell adhesion molecules as well as a greater enrichment of tumor-infiltrating lymphocytes (TILs) [44]. Nonetheless, the current understanding of how m6A alteration contributes to the advancement of Luminal B breast cancer, as well as the pathways involved, is limited and calls for more research.

The DYNLT1, also known as the TCTEX1 gene, has been reported to encode for one of the components of the cytoplasmic dynein, a motor complex that facilitates intracellular transport along microtubules within cells [21]. Emerging evidence points to the possible involvement of DYNLT1 in the onset and progression of cancer. DYNLT1 mediates critical biological activities of glioblastoma cells, among them, proliferation and invasiveness, potentially through the RB phosphorylation and MMP2 secretion, respectively, and independently functions as a prognostic biological marker in glioblastoma [9]. Moreover, DYNLT1 mediates the activation of the REIC/DKK-3 protein and hinders SGTA dimerization, thus promoting the dynein-dependent AR transportation and activating AR signaling in the human prostate cancer cells [33]. Notably, DYNLT1 was upregulated in breast cancer, predicted poor clinical outcome, and was associated with cell proliferation, migration, and metastasis [30]. Additionally, DYNLT1 inhibits Parkin-mediated ubiquitination and degradation of VDAC1, which fuels metabolic processes in mitochondria and the development of breast cancer [19]. However, none of the studies have investigated the involvement of DYNLT1 in Luminal B breast cancer endocrine resistance.

This study's findings showed that Luminal B breast cancer patients recorded significantly overexpressed DYNLT1 levels, which was associated with a poor response to tamoxifen treatment. Importantly, the enhanced expression of DYNLT1 led to ligand-independent activation of the ER signaling pathway and functionally induced a tamoxifen-resistant phenotype in luminal breast cancer cells. Based on this evidence, DYNLT1 could be a crucial predictive marker for endocrine resistance and a promising therapeutic target against relapsed Luminal B breast cancer.

2.1. Cell culture

Hybri-Care Medium containing 1.5 g/L sodium bicarbonate was used to culture the Breast cancer cell line BT474; DMEM with 10% fetal bovine serum (FBS) used to culture T-47D and MCF7; RPMI 1640 with 10% FBS was used to culture HCC1428 and ZR-75-30; Leibovitz’s L-15 medium with 10% FBS was used to culture MDA-MB-361. All the above cell lines were outsourced from the American Type Culture Collection (ATCC, Manassas, VA, USA). This study used phenol red-free DMEM mixed with 10% charcoal/dextran-treated FBS to culture cells for 4-hydroxytamoxifen (4-OH-TAM, #H6278; Sigma-Aldrich) treatment. For these experiments, cells were maintained at 37℃ in a 5% CO₂ incubator.

2.2. Immunohistochemistry

Overall, 62 paraffin-embedded breast cancer specimens were obtained for Immunohistochemistry (IHC) analysis. The histological diagnosis of these specimens was made at Sun Yat-sen University's Seventh Affiliated Hospital. The use of these clinical materials for the study was approved by the Institutional Research Ethics Committee, and consent was acquired from patients in advance. The paraffin-embedded tissue slices were subjected to IHC examination as directed by the manufacturer utilizing Histostain-Plus Kits from Invitrogen in Carlsbad, CA. In addition, IHC staining was conducted with anti-DYNLT1 (Tctex1) rabbit antibody (sc-365567; Santa Cruz Biotechnology, Inc.) and anti-Ki67 mouse monoclonal antibody (1:800 dilution; CST, Shanghai, China). Quantitative analysis of the IHC score was performed using the AxioVision Rel.4.6 computerized image analysis system, with the use of an automated measurement program (Carl Zeiss, Oberkochen, Germany).

2.3. Luciferase activity assay

The 3×ERE (estrogen-responsive element) TATA luc luciferase reporters were acquired from Addgene (plasmid #11354). Next, triplicate 48-well plates were used to culture 3×10³ cells for 24 hours. We subsequently utilized the Lipofectamine 3000 reagent (#L3000015; Invitrogen) to transfect cells with 100 ng of luciferase reporter plasmids or the control luciferase plasmid, together with 1 ng of pRL-TK Renilla plasmid (#E2231; Promega) as directed by the manufacturer. Twenty-four hours following transfection, the Dual-Luciferase Reporter Assay Kit (#E1960; Promega) was utilized to measure the luciferase and Renilla signals.

2.4. RNA stability assay

A 6-well plate was used to culture the breast cancer cells throughout the night. The breast cancer cells were then treated with actinomycin-D (5 µg/ml, MedChemExpress) for the specified time to block gene transcription. Afterward, RT-qPCR was used to ascertain the RNA concentration. A normalization to GAPDH was performed on the RNA levels measured at different points in time in the indicated group.

2.5. RNA immunoprecipitation (RIP) assay

The Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Burlington, MA, USA) was utilized to conduct the RIP assay as indicated in a previous publication [25]. Succinctly, a RIP lysis buffer was utilized to lyse the cells, and magnetic beads were conjugated with a human antibody against ELAVL1/HuR (Cell Signaling Technology) with normal mouse IgG antibody (Millipore, Burlington, MA, USA) as a negative control. Subsequently, the obtained RNA was subjected to RT-qPCR and normalized to the input.

2.6. In vivo experiments

GemPharmatech (Guangdong, China) supplied female BALB/c-nu mice (18–20 g, aged 5 weeks), which were kept on a 12-h light/dark cycle in barrier facilities. Random groupings of mice were established (n = 6/group). Female mice were administered injections of 5 × 10⁶ cells of breast cancer, either expressing DYNLT1 or a control shRNA, into the mammary fat pads to study how DYNLT1 affects tamoxifen resistance in breast tumors, and the sizes of the tumors were recorded at various time points. When the xenografts reached a size of about 200 mm³, tamoxifen citrate (5 mg/pellet; 60-day release, #SE‐351; Innovative Research of America) treatment was started. Every three days, the volume of the tumor was recorded to provide an approximation of the kinetics of the formation of the tumor. The volume of the tumor was determined as follows: (Length*Width²)/2. Then, weight measurements were taken after excising the tumors 4 weeks later. Following tumor dissection, IHC examination was carried out on tumor sections obtained from three separate xenografted animals. Subsequently, the proliferative index was quantified by determining the percentage of Ki67-positive cells. Quantification of the apoptotic index was done by determining the proportion of cells that were stained with the DeadEndTM Fluorometric TUNEL System (#G3250; Promega). Sun Yat-Sen University's Institutional Animal Care and Use Committee approved all the experiments involving animals, and the study followed all the applicable rules and regulations.

2.7. Statistics

GraphPad Prism 9 was utilized for statistical analyses. The comparative analyses for two groups were implemented by calculating the P-values with a Student's t-test For comparison among more than two groups, P-values were calculated using ANOVA test. P < 0.05 was determined to be the significance criterion in all cases.

3.1. The expression levels of DYNLT1 are significantly upregulated in Luminal B breast cancer

The RNA seq transcriptomic data from the Breast Cancer Gene-Expression Miner v5.0 (bc-GenExMiner v5.0) database [20] was analyzed to determine the DYNLT1 expression in breast cancer. Notably, we found that DYNLT1 presented a remarkable upward trend in healthy, tumor-adjacent, and tumor tissues, revealing a significant connection between DYNLT1 expression and tumorigenesis of breast cancer (Fig. 1A). In comparison to the basal-like, HER2-enriched, Luminal A, and normal breast-like subtypes, the Luminal B subtype had considerably upregulated levels of DYNLT1 (Fig. 1B). The expression of DYNLT1 was further confirmed through IHC analysis of 62 paraffin-embedded breast cancer specimens using ERα, PR, HER2, and Ki67 statuses for tumor classification [11]. Consistently, DYNLT1 was substantially upregulated in Luminal B breast cancer tissues when compared to Luminal A, HER2-enriched, and triple-negative breast cancer (TNBC) subtypes (Fig. 1C-E). Collectively, our results demonstrated an upregulation of DYNLT1 levels specifically in Luminal B breast cancer.

3.2. The high expression of DYNLT1 is indicative of an unfavorable clinical recurrence-free survival (RFS) outcome in Luminal B breast cancer patients undergoing tamoxifen therapy

To determine how the DYNLT1 expression levels were related to the survival of breast cancer patients, the online tool, namely the Kaplan–Meier plotter (KM) [12], was adopted to evaluate the clinical significance of DYNLT1 in breast cancer. Notably, it was revealed that DYNLT1 upregulation was correlated with dismal RFS in all breast cancer cases (Hazard Ratio (HR) = 1.24, P = 0.00023) (Supplemental Fig. 1A). Next, we explored the RFS in breast cancer patients stratified with distinct molecular subtypes. The KM analysis indicated that DYNLT1 expression upregulation was significantly related to unfavorable RFS in Luminal B (HR = 1.53, P = 0.00014), Luminal A (HR = 1.3, P = 0.002), and HER2-enriched (HR = 1.56, P = 0.014) breast cancer subtypes, but favorable in the basal-like subtype (HR = 0.76, P = 0.027) (Supplemental Fig. 1B).

Luminal breast cancer is treated with tamoxifen as a first-line drug, but the drug resistance is often inescapable. We further explored whether the expression of DYNLT1 is related to the effectiveness of endocrine therapy in Luminal breast cancer. Analysis of the KM plotter database indicated that higher DYNLT1 expression significantly linked to unfavorable RFS in all breast cancer cases with tamoxifen-only (HR = 1.47, P = 0.0085) or endocrine-include (HR = 1.29, P = 0.047) therapy, but not in endocrine-exclude cases (HR = 0.87, P = 0.083) (Fig. 2A). Interestingly and importantly, when we performed subtype-specific survival analysis, the DYNLT1 expression upregulation was significantly linked to dismal RFS in Luminal B with tamoxifen-only (HR = 2.23, P = 0.00084) or endocrine-include therapy (HR = 1.6, P = 0.0094), and Luminal A with tamoxifen-only (HR = 1.51, P = 0.048), but not in Luminal A with endocrine-include (HR = 1.3, P = 0.12) and other endocrine-exclude breast cancer cases (Luminal B, HR = 1.35, P = 0.064; Luminal A, HR = 0.84, P = 0.29) (Fig. 2B and C). Therefore, we conclude that DYNLT1 expression upregulation is correlated with the relapse of Luminal B breast cancer treated with tamoxifen, resulting in poorer clinical outcomes.

3.3. Elevated DYNLT1 expression induces resistance to tamoxifen both in vitro and in vivo

The DYNLT1 expression in the luminal breast cancer cell lines was analyzed to ascertain the possible involvement of DYNLT1 in tamoxifen resistance. Notably, DYNLT1 showed significantly upregulation in Luminal B breast cancer cell lines (ZR-75-30, BT-474, and MDA-MB-361) relative to Luminal A cells (MCF7, T47D, and HCC1428) (Fig. 3A). We searched for the IC50 value of tamoxifen in these cell lines from Genomics of Drug Sensitivity in Cancer (GDSC) [45] to investigate how DYNLT1 expression was correlated with drug sensitivity in breast cancer. Surprisingly, elevated DYNLT1 expression was associated with drug resistance to tamoxifen (R = 0.855, P = 0.030) (Fig. 3B). Subsequently, we analyzed RNA seq data from the Gene Expression Omnibus (GEO) dataset (GSE106681) [5], comparing the expression levels of DYNLT1 in MCF7 and its tamoxifen-sensitive but aggressive subline (WS8) as well as tamoxifen-resistant MCF7-WS8 subline (TAMR). Remarkably, our results revealed significantly elevated DYNLT1 levels in TAMR cells relative to those in WS8 or parental MCF7 cells, regardless of tamoxifen treatment (Fig. 3C).

Our tamoxifen-resistant MCF7-TMR cell line, which originated from the tamoxifen-sensitive Luminal A breast cancer cell line MCF7, was tested for DYNLT1 expression to provide additional confirmation of the aforementioned findings [46]. In comparison to parental MCF7 cells, MCF7-TMR cells exhibited a markedly elevated DYNLT1 expression level (Fig. 3D). We established ZR-75-30 and MCF7-TMR cell lines, stably transduced with a DYNLT1 shRNA, as well as MCF7, stably expressing DYNLT1, to study the biological function of DYNLT1 and ascertain its involvement in tamoxifen resistance progression (Fig. 3E). Then, tamoxifen’s cytotoxic activity was explored on breast cancer cell lines. DYNLT1 silencing markedly increased the sensitivity of tamoxifen in ZR-75-30 cells making IC₅₀ shift from 168.655 to 29.376 or 31.989 µM and MCF7-TMR (IC₅₀, 41.591 to 14.588 or 16.982) cells, while DYNLT1 overexpression decreased the sensitivity of tamoxifen in MCF7 (IC₅₀, 11.324 to 35.237) (Fig. 3F). The results show that the DYNLT1 overexpression induces a tamoxifen-resistant phenotype in luminal breast cancer cells.

Thereafter, the impact of DYNLT1 overexpression on tamoxifen resistance in breast cancer was assessed in vivo. One week following the implantation of E2 pellets, female mice had their mammary fat pads injected with either control cells or MCF7/DYNLT1 cells, and the first dosage of tamoxifen was administered after the tumor volume was about 200 mm³. The anti-tumor impact of tamoxifen therapy was considerably diminished in the MCF7 xenografts when DYNLT1 was overexpressed, as illustrated in Fig. 4A-C. Additionally, the apoptotic rates were lower and Ki67 levels were greater in tamoxifen-treated MCF7/DYNLT1 xenografts in comparison to control tumors (Fig. 4D and E), which supports the hypothesis that upregulation of DYNLT1 confers tamoxifen-resistant tumorigenicity to Luminal B breast cancer cells. Furthermore, Mice were administered injections with either control cells or MCF7-TMR/DYNLT1-Ri cells into their mammary fat pads, and treatment with tamoxifen commenced when the lesions had attained an approximate measurement of 200 mm³. Notably, Fig. 4F and G demonstrates that DYNLT1 knockdown in the MCF7-TMR xenografts without tamoxifen treatment significantly reduced the tumor size and weight, while co-therapy with tamoxifen and DYNLT1 shRNA more effectively blocked the tumorigenic potential of MCF7-TMR cells. In comparison to control tumors, tamoxifen-treated MCF7-TMR/DYNLT1-Ri xenografts consistently exhibited a more pronounced decrease in Ki67 levels and elevated apoptotic rates (Fig. 4H and I). These results suggest that silencing DYNLT1 in tamoxifen-resistant breast cancer cells makes them more responsive to the drug.

3.4. Overexpression of DYNLT1 facilitates ligand-independent activation of the ER signaling pathway

We first used GPS-pro to determine proteins potentially interacting with DYNLT1 to probe the molecular mechanisms via which DYNLT1 leads to tamoxifen resistance (Fig. 5A). The ingenuity pathway analysis g:Profiler [22] was utilized to analyze DYNLT1-interacting proteins enriched pathways. The results revealed that DYNLT1-interacting proteins exhibited significant enrichment in Gene Ontology Molecular Functions, including binding to dynein intermediate/heavy chain, identical protein binding, and microtubule motor activity (Fig. 5B). In terms of Biological Processes, enrichment was observed in microtubule-based processes, cell division, and the cell cycle. Additionally, these proteins were found to be localized in Cellular Components such as the cytoplasmic dynein complex, microtubules, and the nucleoplasm. These biological functions are recognized to have an essential function in the development of resistance to cancer therapies. Subsequently, Gene Set Enrichment Analysis (GSEA) was executed utilizing the online Webgestalt tool [26] to analyze genes coexpressed with DYNLT1 from cBioPortal [10], using data from The Cancer Genome Atlas (TCGA) Breast invasive carcinoma (BRCA). We discovered that hormone-mediated signaling pathway activity correlates positively with DYNLT1 expression (Fig. 5C), indicating that DYNLT1 upregulation may have a key function in ER signaling pathway activation.

We designed an estrogen-responsive element (ERE)-luciferase reporter to more thoroughly investigate whether DYNLT1 controls ERα transcriptional activity. Notably, overexpression of DYNLT1 in MCF7 cells ligand-dependently enhanced ERα transcriptional activity, whereas silencing DYNLT1 restored MCF7-TMR cell sensitivity to estrogen and tamoxifen (Fig. 5D). Additionally, the expression of trefoil factor 1 (TFF1) and progesterone receptor (PGR), known as classical ERα target genes, exhibited a significant increase in DYNLT1-overexpressing MCF7 cells, which were reduced in DYNLT1-silenced MCF7-TMR cells without estrogen or tamoxifen treatment (Fig. 5E). Although DYNLT1-overexpressed MCF7 cells were unaffected by tamoxifen treatment, DYNLT1-silenced MCF7-TMR cells showed a stronger inhibition of gene expression (Fig. 5E). The findings confirm that the ER-dependent transcriptional network is functionally regulated by DYNLT1.

3.5 ELAVL1-mediated m6A modification contributes to the upregulation of DYNLT1 expression and facilitates the acquisition of tamoxifen resistance phenotype

The most prevalent type of RNA epigenetic modification in higher eukaryotic mRNAs, m6A, is demonstrated to be critically involved in several types of biological processes [7, 13, 15, 49]. Firstly, SRAMP [50] was employed to predict the occurrence of m6A modification sites on the DYNLT1 RNA sequences. Two m6A sites with high confidence and one m6A site with moderate confidence across the mRNA transcript of DYNLT1 were identified (Fig. 6A). According to the m6A RIP test, the m6A level was higher in the ZR-75-30 and MCF7-TMR cells in comparison to MCF7, indicating that m6A may be involved in DYNLT1 upregulation (Fig. 6B). To find out which m6A regulators are implicated in regulating DYNLT1 expression, the link between the expression levels of writers, erasers, and readers (WERs) of m6A and DYNLT1 was further assessed using cBioPortal in the breast cancer tissue. The results revealed that the ELAVL1, WTAP, METTL5, and HNRNPC expression was positively linked with the DYNLT1 expression (P < 0.001, Spearman R > 0.3) (Fig. 6C, and Supplemental Fig. 2A). We then used ENCORI [24] to predict the interaction between DYNLT1 mRNA and the corresponding RNA binding proteins (RBPs), showing that DYNLT1 mRNA had a possible interaction with ELAVL1 and HNRNPC (Supplemental Fig. 2B). Further, we analyzed the iCLIP-seq data conducted in MCF7 cells from the ENCORI database and identified that the ELAVL1 binding site located at chr6:158636709–158636748[-] on Exon5/5 of DYNLT1 (NM_006519), which precisely corresponded to the #3 m6A site predicted by SRAMP (Fig. 6D). As expected, RIP analysis demonstrated that DYNLT1 mRNA was highly enriched in ELAVL1-immunoprecipitated complexes, compared to the IgG control group (Fig. 6E).

Reports indicate that ELAVL1, a critical component of the N6-methyltransferase complex, improves mRNA stability in breast cancer cells that are insensitive to tamoxifen [39]. Next, we used siRNA to target ELAVL1 in breast cancer cells. In both the ZR-75-30 and MCF7-TMR cell lines, we observed a significant drop in m6A and DYNLT1 mRNA levels after ELAVL1 silencing (Fig. 6F and G). After that, we investigated how ELAVL1 siRNA affected the stability of DYNLT1 mRNA and discovered that breast cancer cells treated with this siRNA had a much shorter half-life of DYNLT1 mRNA (Fig. 6H and I), supporting the notion that ELAVL1 controlled DYNLT1 expression by influencing the stability of DYNLT1 mRNA. Overall, the above evidence confirms that silencing of ELAVL1 attenuates the m6A modification of DYNLT1 mRNA and subsequently reduces the expression of DYNLT1.

Furthermore, KM analysis indicated that ELAVL1 expression upregulation significantly related to worse RFS in all or Luminal B breast cancer cases with endocrine-include therapy (all, HR = 1.92, P = 3.2e-09; Luminal B, HR = 1.75, P = 0.00083), but not in endocrine-exclude cases (all, HR = 0.86, P = 0.081; Luminal B, HR = 1.24, P = 0.15) (Supplemental Fig. 3A and B). In Luminal B breast cancer with tamoxifen-only therapy, although the trend of the survival curve is consistent with the above conclusions, the sample size was not sufficient to attain statistical significance (HR = 1.61, P = 0.065) (Supplemental Fig. 3B). Thus, high levels of ELAVL1 predict poor clinical RFS in endocrine-treated breast cancer patients.

Moreover, we investigated how ELAVL1 affected tamoxifen resistance in parental MCF7 and MCF7-TMR breast cancer cell lines. Notably, ELAVL1 expression upregulation significantly attenuated the anti-tumor effect of tamoxifen treatment in MCF7 cells, while DYNLT1 silencing resensitized ELAVL1 overexpressing-MCF7 cells to tamoxifen treatment (Fig. 6J). Conversely, the knockdown of ELAVL1 restored sensitivity to tamoxifen treatment in MCF7-TMR cells, whereas DYNLT1 expression upregulation significantly abrogated the anti-tumor properties of ELAVL1 silencing on MCF7-TMR cells under tamoxifen therapy (Fig. 6K). Collectively, these data suggest that upregulation of ELAVL1 facilitates m6A modification in DYNLT1 mRNA and subsequently augments DYNLT1 expression, thereby promoting tamoxifen resistance in Luminal B breast cancer.

Most patients with hormone receptor-positive breast cancer typically receive endocrine therapy as the first-line treatment, which forms the foundation of drug therapy. According to a meta-analysis, invasive breast cancer patients who underwent five years of treatment with selective estrogen receptor modulators (SERMs) experienced an absolute risk reduction that was approximately nine times greater than that of those who did not receive treatment [38]. Nevertheless, resistance is an emerging challenge with endocrine therapy for breast cancer and is often caused by ER mutations, CCND1 and CDK6 gene overexpression, and PIK3CA gene alterations [17, 37, 41]. Therefore, a thorough assessment of the factors that influence patient outcomes and identification of potential biomarkers in endocrine-resistant breast cancer will help to formulate the best individualized treatment plan for breast cancer patients. Interestingly, our findings highlighted that Luminal B subtype breast cancer is characterized by significantly elevated DYNLT1 levels. Moreover, analysis of the prognostic database indicated that clinical RFS was worse for tamoxifen-treated individuals with luminal B breast cancer who expressed DYNLT1 at high levels. In patients with Luminal B breast cancer, our findings point to DYNLT1 as a potential new biological marker for prognosis prediction.

After tamoxifen therapy, individuals with Luminal A-like breast cancer experience a more favorable prognosis than those with Luminal B-like disease [29]. However, when exposed to the HER3 ligand heregulin (HRG), Luminal A MCF7 breast cancer cells, which are normally highly responsive to tamoxifen, can overcome tamoxifen's anti-proliferative impacts. This is because the cells activate a proliferative signature comparable to that of Luminal B-type tumors, which are insensitive to tamoxifen [27]. Research into finding important molecular targets that might be therapeutically beneficial in tamoxifen-refractory Luminal B breast cancer is still in its nascent stages, beyond the anticipated hyperactivation of downstream signaling cascades including ERK/MAPK and PI3K/AKT [3, 42]. Our findings demonstrated an upregulation of DYNLT1 in Luminal B breast cancer cases that were resistant to endocrine treatment, and DYNLT1 has potential as a predictive marker for tamoxifen resistance. Furthermore, we verified that overexpression of DYNLT1 in the MCF7 cell lines/xenografts significantly impaired the anti-tumor effect of tamoxifen treatment, while silencing DYNLT1 increased sensitivity of MCF7-TMR tamoxifen-resistant breast tumors to tamoxifen therapy. In addition, activation of ER-dependent transcriptional network mediated by high DYNLT1 levels could facilitate the advancement of Luminal B breast cancer to endocrine-resistant state. Co-therapy with tamoxifen and DYNLT1 inhibition could be useful in overcoming tamoxifen resistance in Luminal B breast cancer, nevertheless, this hypothesis requires more clinical trials to be validated.

m6A methylation, akin to DNA methylation, is regulated by methyltransferase and demethylase enzymes. However, it modulates the post-transcriptional expression level of a gene without changing the base sequence. The epigenetic modification of m6A methylation can affect the occurrence and advancement of cancers by modulating the expression levels of oncogene and tumor suppressor gene mRNA molecules [18]. Previous studies have revealed that increased m6A reader YTHDF1 and YTHDF3 abnormalities in breast cancer tissues links to metastatic disease and a worse prognosis for patients with this malignancy [2]. Hsa-miR-146a-5p overexpression modulated by METTL14 induced-m6A levels increase promotes the migratory rate and invasiness of MDA-MB-231 and MCF-7cells [47]. Under hypoxic conditions, ALKBH5-mediated m6A-demethylation of NANOG mRNA leads to an upregulation in both NANOG mRNA and protein expression, thus enhancing the acquisition of breast cancer stem cell characteristics and enhancing their tumorigenic potential [48]. Our study reveals that the m6A reader ELAVL1/HuR promoted the m6A modification in DYNLT1 mRNA and increased the expression levels of DYNLT1. The accumulation of the RNA binding protein ELAVL1/HuR in the cytoplasm is crucial for estrogen receptor-positive breast cancer cells to develop tamoxifen resistance, given that it induces an acute response to tamoxifen by enhancing the stability of mRNA transcripts that encode for drug-resistant transcripts [14]. Tamoxifen resistance is conferred on estrogen receptor-positive breast cancer cells through HuR-mediated enhancement of ERBB2 mRNA stability and translation [39]. The advancement of cancer has been reported to be impeded by various HuR inhibitors, such as N-Benzylcantharidinamide, MS-444, and KH-3 [8, 23, 31]. Of these, KH-3 was demonstrated to interfere with the HuR-FOXQ mRNA interaction, thereby suppressing the proliferative rate and metastatic spread of breast cancer cells [43]. Thus, the HuR-dependent DYNLT1 could be a therapeutic target for endocrine-resistant breast cancer; however, this hypothesis necessitates further in vivo evidence as well as the specific processes that are dependent on HuR.

In summary, this study demonstrated the high expression of DYNLT1 in Luminal B breast cancer and predicted a greater risk of relapse and unfavorable clinical RFS after tamoxifen therapy. Mechanistically, m6A methylation-mediated DYNLT1 upregulation by ELAVL1 facilitates tamoxifen resistance in Luminal B breast cancer by increasing the ligand-independent activation of the ER signaling pathway. To avoid Luminal B breast cancer patients developing tamoxifen resistance, we suggest a treatment approach focused on suppressing DYNLT1 and/or ELAVL1.

Data availability statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Funding

This work was supported by grants from the National Natural Science Foundation of China (82072905, 82372907), Guangdong Basic and Applied Basic Research Foundation (2022A1515011111, 2023A1515030058), Shenzhen Science and Technology Program (RCYX20210706092141082, JCYJ20210324123011031, JCYJ20210324121809026, JCYJ20230807110701003), Research start-up fund of part-time PI, SAHSYSU (ZSQYJZPI202008).

Competing interests

The authors declare no conflict of interest in performing this study.

Author Contributions

Qiji Li, Chenxin Li, and Di Zhang designed the experiments and were responsible for all data collection and analysis. Kefeng Lei and Yun Wang collected clinical tissues. Qingqing Zhu and Yuhao Zhang performed, cellular experiments and biochemical experiments. Xiaoting Sun, Zihan Zheng and Xiaoying Yang conducted the animal experiments. Qin Tian, Chengming Zhu and Liping Ye wrote the manuscript and supervised the project. All authors read and approved the manuscript.

Ethics statement

The use of these clinical materials for the study was approved by the Institutional Research Ethics Committee of Sun Yat-sen University's Seventh Affiliated Hospital, and consent was acquired from patients in advance. Sun Yat-Sen University's Institutional Animal Care and Use Committee approved all the experiments involving animals, and the study followed all the applicable rules and regulations.

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N6-methyladenosine modification of DYNLT1 Facilitates Tamoxifen Resistance in Luminal B Breast Cancer

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Abstract

Purpose

Methods

Results

Conclusion

Figures

1. Introduction

2. Materials and methods

2.1. Cell culture

2.2. Immunohistochemistry

2.3. Luciferase activity assay

2.4. RNA stability assay

2.5. RNA immunoprecipitation (RIP) assay

2.6. In vivo experiments

2.7. Statistics

3. Results

3.1. The expression levels of DYNLT1 are significantly upregulated in Luminal B breast cancer

3.3. Elevated DYNLT1 expression induces resistance to tamoxifen both in vitro and in vivo

3.4. Overexpression of DYNLT1 facilitates ligand-independent activation of the ER signaling pathway

4. Discussion

5. Conclusions

Declarations

References

Additional Declarations

Supplementary Files

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Version 1