Role of

doi:10.3969/j.issn.1674-8115.2025.10.005

Abstract

Abstract:

Objective ·To determine hyaluronan (HA) expression in the endocrine-resistant microenvironment of estrogen receptor-positive (ER⁺) breast cancer and elucidate its impact on the acquired resistance. Methods ·Chemiluminescent immunoassay was used to quantify HA levels in the culture supernatants of fulvestrant-resistant breast cancer cells. An immunofluorescence (IF) assay was performed to visualize the colocalization of CD44 and HA in MCF7/FulR cells. Using an established adaptive endocrine-resistant breast cancer mouse model, HA expression in resistant breast cancer tissues was assessed by immunohistochemistry (IHC) assay. Single-cell RNA sequencing (scRNA-seq) and RNA sequencing (RNA-seq) were conducted to examine transcriptomic profiles and alterations in HA-related genes in resistant breast cancer cells. Flow cytometry (FCM) was utilized to measure the proportion of CD44⁺CD24^- cells in MCF7/FulR. The correlation between HA synthesis genes and cell stemness was investigated in clinical ER⁺ breast cancers from GEO data sets. Hyaluronidase (HAase) treatment was applied to remove the "HA coat", and RT-qPCR and Western blotting analysis were carried out to monitor changes in stemness-related molecules. CCK-8 assays, flow cytometry (FCM), and Hoechst 33258 staining were performed to determine changes in apoptosis and fulvestrant efficiency after HAase treatment. Results ·IF results revealed that compared with MCF7 cells, the "HA coat" on the surface of MCF7/FulR cells was significantly thickened. IHC demonstrated markedly increased HA retention in fulvestrant-resistant mouse breast cancer tissues. ScRNA-seq and RNA-seq analyses indicated elevated expression of stemness-related genes and HA synthesis-associated genes in fulvestrant-resistant breast cancer cells. Correlation analysis revealed a positive association between HA synthesis and cancer stemness in ER⁺ breast cancer. IF and RT-qPCR results demonstrated that removing the HA coating from the surface of MCF7/FulR cells led to a significant reduction in the expression of stemness-related molecules; concurrently, CCK-8 assays, FCM analysis, and Hoechst 33258 staining revealed that "HA coat" clearance reduced MCF7/FulR' tolerance to fulvestrant and increased apoptosis. Conclusion ·Endocrine-resistant breast cancer cells develop an enriched "HA coat", which promotes stemness in fulvestrant-resistant tumors. Disruption of this HA coat through HAase treatment effectively reduces cell stemness, induces apoptosis, and re-sensitizes breast cancer cells to fulvestrant.

Key words: breast cancer, endocrine therapy-resistant tumor, hyaluronan coat

CLC Number:

R737.9

WU Shiyi, CHEN Si, LIU Bohan, LIU Yuting, LIU Yiwen, HE Yiqing, DU Yan, ZHANG Guoliang, GUO Qian, GAO Feng, YANG Cuixia. Role of "HA coat" in modulating stemness and endocrine resistance in ER⁺ breast cancer[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(10): 1298-1307.

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Gene	Primer sequence
KLF4	Forward	5'-CATCTCAAGGCACACCTGCGAA-3'
	Reverse	5'-TCGGTCGCATTTTTGGCACTGG-3'
SNAIL	Forward	5'-TGCCCTCAAGATGCACATCCGA-3'
	Reverse	5'-GGGACAGGAGAAGGGCTTCTC-3'
SOX2	Forward	5'-GCTACAGCATGATGCAGGACCA-3'
	Reverse	5'-TCTGCGAGCTGGTCATGGAGTT-3'
SOX9	Forward	5'-AGGAAGCTCGCGGACCAGTAC-3'
	Reverse	5'-GGTGGTCCTTCTTGTGCTGCAC-3'
TWIST	Forward	5'-GCCAGGTACATCGACTTCCTCT-3'
	Reverse	5'-TCCATCCTCCAGACCGAGAAGG-3'
VIM	Forward	5'-AGGCAAAGCAGGAGTCCACTGA-3'
	Reverse	5'-ATCTGGCGTTCCAGGGACTCAT-3'
ZEB1	Forward	5'-GGCATACACCTACTCAACTACGG-3'
	Reverse	5'-TGGGCGGTGTAGAATCAGAGTC-3'
NANOG	Forward	5'-CTCCAACATCCTGAACCTCAGC-3'
	Reverse	5'-CGTCACACCATTGCTATTCTTCG-3'
ACTB	Forward	5'-CCCGAGCTGATTGACTACGG-3'
	Reverse	5'-GCAGTTCCAACGATGTCTTCG-3'

Gene	Primer sequence
KLF4	Forward	5'-CATCTCAAGGCACACCTGCGAA-3'
	Reverse	5'-TCGGTCGCATTTTTGGCACTGG-3'
SNAIL	Forward	5'-TGCCCTCAAGATGCACATCCGA-3'
	Reverse	5'-GGGACAGGAGAAGGGCTTCTC-3'
SOX2	Forward	5'-GCTACAGCATGATGCAGGACCA-3'
	Reverse	5'-TCTGCGAGCTGGTCATGGAGTT-3'
SOX9	Forward	5'-AGGAAGCTCGCGGACCAGTAC-3'
	Reverse	5'-GGTGGTCCTTCTTGTGCTGCAC-3'
TWIST	Forward	5'-GCCAGGTACATCGACTTCCTCT-3'
	Reverse	5'-TCCATCCTCCAGACCGAGAAGG-3'
VIM	Forward	5'-AGGCAAAGCAGGAGTCCACTGA-3'
	Reverse	5'-ATCTGGCGTTCCAGGGACTCAT-3'
ZEB1	Forward	5'-GGCATACACCTACTCAACTACGG-3'
	Reverse	5'-TGGGCGGTGTAGAATCAGAGTC-3'
NANOG	Forward	5'-CTCCAACATCCTGAACCTCAGC-3'
	Reverse	5'-CGTCACACCATTGCTATTCTTCG-3'
ACTB	Forward	5'-CCCGAGCTGATTGACTACGG-3'
	Reverse	5'-GCAGTTCCAACGATGTCTTCG-3'

Role of "HA coat" in modulating stemness and endocrine resistance in ER⁺ breast cancer

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Role of "HA coat" in modulating stemness and endocrine resistance in ER+ breast cancer

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Role of "HA coat" in modulating stemness and endocrine resistance in ER⁺ breast cancer