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. 2026 Apr 29;152(4):101. doi: 10.1007/s00432-026-06477-x

HDHD5 promotes triple-negative breast cancer growth and drives EMT-associated phenotypes via regulating S100A4

Jia-Yang Cai 1,2,✉, Yin-Ling Zhang 3, Fang-Lin Zhang 3, Qian Zhao 1,2,3, Meng-Ke Ma 1,2,3, Zhi-Min Shao 1,2,3,✉
PMCID: PMC13129178  PMID: 42056314

Abstract

Purpose

Triple-negative breast cancer (TNBC) displays an aggressive clinicopathological profile. Despite cytotoxic chemotherapy being the primary systemic therapy, complete responses are achieved in fewer than 30% of patients, highlighting the need for new therapeutic targets and a deeper understanding of disease-driving mechanisms.

Methods

This study identified key oncogenic factors in TNBC through multi-omics differential analyses. Cell proliferation, migration, and invasion abilities were assessed using CCK-8, colony formation, and transwell migration and invasion assays. Quantitative proteomics was applied to profile downstream protein alterations following HDHD5 knockdown. Western blotting and RT-qPCR were used to examine expression levels in different samples. Xenograft tumor model was employed to investigate the in vivo functions of HDHD5.

Results

HDHD5 was highly expressed in TNBC and its elevated expression was associated with poor patient prognosis. Functional studies demonstrated that HDHD5 promotes TNBC cell proliferation and colony formation, as well as enhances cell migration and invasion. Mechanistically, HDHD5 facilitates epithelial–mesenchymal transition (EMT) associated phenotypes and promotes migratory and invasive capacities in TNBC cell lines, at least in part, through regulation of S100A4. Consistently, HDHD5 promoted TNBC tumor growth both in vitro and in vivo via S100A4.

Conclusion

Our study reveals HDHD5 as a previously unrecognized driver of tumor progression in TNBC and highlights its potential as a therapeutic target and prognostic biomarker for this aggressive breast cancer subtype.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00432-026-06477-x.

Keywords: HDHD5, Triple-negative breast cancer, S100A4, EMT, Migration, Invasion

Introduction

Triple-negative breast cancer (TNBC) represents a distinct subtype of breast cancer that lacks expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, and it comprises roughly 15% of all breast cancer diagnoses (Bianchini et al. 2022). In contrast to other breast cancer subtypes, triple-negative breast cancer is characterized by highly aggressive clinicopathological features, including early distant metastasis, frequent relapse, and poor clinical outcomes (Bianchini et al. 2016). Because triple-negative breast cancer lacks responsiveness to endocrine therapy and HER2-targeted agents, cytotoxic chemotherapy remains the cornerstone of systemic treatment (Bianchini et al. 2022, Bianchini et al. 2016). However, fewer than 30% of patients achieve a complete response to standard chemotherapy, highlighting the limited effectiveness of current therapeutic options (Li et al. 2022). Therefore, a deeper understanding of the molecular mechanisms underlying triple-negative breast cancer, together with the identification of novel therapeutic targets based on its molecular characteristics, is urgently needed.

Mitochondria play a central role in cellular energy production and are essential for key processes including cell signaling, metabolism, apoptosis, and calcium homeostasis (Nunnari and Suomalainen 2012). In tumors, mitochondria also play essential roles in regulating cell growth, cell death, and cellular metabolism throughout all stages of tumor progression (Tanprasert et al. 2022). In recent years, mitochondrial-related genes have been extensively investigated in breast cancer. Xiong Z et al. demonstrated that PRKN-mediated regulation of mitophagy promotes tumor growth and metastasis in breast cancer (Xiong et al. 2025). In addition, multiple studies have shown that prognostic models based on mitochondrial-related gene signatures can effectively predict clinical outcomes in patients with breast cancer (Xu et al. 2025, Ding et al. 2024, Yu et al. 2025). Integrative analysis of our previously published multi-omics datasets from FUSCC-TNBC (Jiang et al. 2019) and CBCGA (Jiang et al. 2024) identified the mitochondrial-related gene Haloacid dehalogenase–like hydrolase domain–containing 5 (HDHD5, also known as CECR5) as being markedly upregulated in triple-negative breast cancer tissues relative to matched adjacent normal tissues. Moreover, elevated HDHD5 expression was strongly associated with unfavorable clinical outcomes in patients with TNBC. HDHD5 has been reported to localize to mitochondria and to be closely involved in the regulation of mitochondrial function and lipid metabolism (Jiang et al. 2022). Recent studies have shown that HDHD5 is highly expressed in colorectal cancer, and that HDHD5-associated gene signatures can predict patient prognosis and immune status (Wang et al. 2025). However, the functional role and mechanistic actions of HDHD5 in tumor biology remain largely undefined, and its involvement in breast cancer has not yet been elucidated. These gaps underscore the need for a comprehensive investigation into the biological functions and underlying mechanisms of HDHD5 in breast cancer.

In this study, leveraging large-scale clinical cohorts previously established in FUSCC, we systematically screened mitochondrial-related genes and identified HDHD5 as markedly upregulated in triple-negative breast cancer. HDHD5 expression was significantly higher in TNBC than in other breast cancer subtypes, and elevated HDHD5 levels were associated with poor patient prognosis. Functional analyses revealed that HDHD5 promotes TNBC cell proliferation, migration, and invasion. Mechanistically, HDHD5 enhanced the malignant behavior of TNBC cells by upregulating S100 Calcium Binding Protein A4 (S100A4) while driving the EMT-associated phenotypes. Moreover, in vivo studies demonstrated that HDHD5 accelerated tumor growth in a xenograft tumor model. Collectively, these results suggest that HDHD5 represents a promising therapeutic target and a potential prognostic biomarker for triple-negative breast cancer.

Materials and methods

Cell culture and chemicals

Human mammary epithelial cells (HMECs), human embryonic kidney 293T cells, and human breast cancer cell lines (LM2-4175, SUM159, MDA-MB-231, MDA-MB-468, MDA-MB-157, Hs578T, BT549, and HCC1806) were obtained from the Shanghai Key Laboratory of Breast Cancer and the Fudan University Shanghai Cancer Center (FUSCC). All cell lines were authenticated by short tandem repeat profiling and routinely monitored for cell viability before experimental use. HMEC cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; BasalMedia, #L110) supplemented with 5% donor horse serum (Gibco, #16050-114), epidermal growth factor (20 ng/mL; Sino Biological, #10605-HNAE), hydrocortisone (0.5 mg/mL; Yeasen, #40109ES08), recombinant human insulin (10 mg/mL; Yeasen, #40107ES76), and 1% penicillin–streptomycin (BasalMedia, #S110B). All other cell lines were maintained in DMEM supplemented with 10% fetal bovine serum (Gibco, #10270-106) and 1% penicillin–streptomycin (BasalMedia, #S110B). Cells were cultured under standard conditions following the suppliers’ recommendations.

Clinical samples and datasets

Human tissue specimens were collected from patients with primary triple-negative breast cancer who underwent surgical treatment at the Fudan University Shanghai Cancer Center. All patients were treatment-naïve at the time of surgery and had not received chemotherapy or radiotherapy. Paired adjacent normal tissues were obtained concurrently and verified by histopathological examination to be free of tumor involvement. This study was conducted in compliance with the ethical principles outlined by the World Medical Association and adhered to the Declaration of Helsinki. Ethical approval was granted by the institutional ethics committee of Fudan University Shanghai Cancer Center. Written informed consent was obtained from all participants prior to surgical intervention.

The multi-omics datasets of triple-negative breast cancer generated at the FUSCC, including RNA sequencing and quantitative proteomics data, together with corresponding clinical information, have been reported previously (Jiang et al. 2019). The Chinese Breast Cancer Genome Atlas (CBCGA) datasets used in this study have also been described elsewhere (Jiang et al. 2024). RNA-seq dataset of TNBC from The Cancer Genome Atlas (TCGA) was downloaded from https://portal.gdc.cancer.gov. Mitochondria-related genes were obtained from the MitoCarta3.0 database (https://www.broadinstitute.org/mitocarta) (Rath et al. 2021). The TIMER2.0 database (http://timer.cistrome.org/) was used to perform a pan-cancer analysis of HDHD5(Li et al. 2020). The Kaplan–Meier Plotter database (https://kmplot.com/) was used to evaluate the prognostic value of HDHD5, with the optimal cutoff value applied (Győrffy 2021).

DNA plasmids construction and transfection

Short hairpin RNA targeting HDHD5 (sequences listed in Supplementary Table S1) was cloned into the pLKO.1 vector. The HA-tagged HDHD5 expression construct was synthesized by Syn-Biotech. The Flag-tagged S100A4 expression vector was generated by GeneChem. Plasmid transfection was performed in HEK293T cells using a Neofect DNA transfection reagent (Tengyi Biol, #TF201201) according to the manufacturer’s instructions. Viral supernatants were harvested 48 h after transfection, passed through a filter, and subsequently applied to target cells in the presence of polybrene (8 µg/mL; Sigma-Aldrich, #H9268) to enhance infection efficiency. Forty-eight hours following infection, stable cell populations were established by antibiotic selection with puromycin (2 µg/mL; Selleck Chemicals, #S7417).

Cell Counting kit-8 (CCK-8) assays

For Cell Counting Kit-8 (CCK-8) assays, cells were seeded into 96-well plates. At the indicated time points, the culture medium was replaced with fresh medium containing CCK-8 reagent (Yeasen Biotechnology, #40203ES92). After incubation for 2 h, absorbance at 450 nm was measured to evaluate cell viability.

Colony formation assays

For colony formation assays, cells were seeded into 12-well plates and maintained for 14 days. Colonies were then fixed with methanol for 1 h and stained with 0.2% crystal violet for 2 h. Images were acquired, and colonies were counted for quantitative analysis.

Antibody and Western blotting

Details of the antibodies used in this study are provided in Supplementary Table S2. For western blot analysis, total protein was isolated from cultured cells using RIPA lysis buffer supplemented with protease and phosphatase inhibitor cocktails (Bimake, #B14002 and #B15003, respectively). Protein concentrations were normalised and equal amounts of lysates were subjected to SDS–PAGE followed by transfer onto PVDF membranes (Merck Millipore, #IPVH00010). After blocking with 5% bovine serum albumin (Sigma-Aldrich, #V900933), membranes were incubated with the indicated primary antibodies and corresponding secondary antibodies. Protein signals were visualized using enhanced chemiluminescence reagents (Tanon, #180-5001E).

RNA extraction and quantitative real-time PCR

Total RNA was isolated from cultured cells with TRIzol reagent (#9109, Takara). Complementary DNA was then synthesized using HiScript III RT SuperMix (#R323-01, Vazyme). Quantitative real-time PCR was carried out with ChamQ Universal SYBR qPCR Master Mix (#Q711-03, Vazyme). Transcript abundance was determined with the 2^−ΔΔCT method. Primer sequences for all qPCR analyses are provided in Supplementary Table S3.

Cell migration and invasion assays

Cell migration and invasion assays were performed using transwell chambers, either uncoated or pre-coated with Matrigel, placed in 24-well plates containing DMEM supplemented with 10% fetal bovine serum in the lower chambers. Cells were suspended in serum-free DMEM and seeded into the upper chambers. After 24 h of incubation, migrated or invaded cells were fixed with methanol and stained with 0.2% crystal violet. Cells on the upper surface of the membranes were removed, and those on the lower surface were imaged and quantified.

Proteomic assays and bioinformatic analyses

To explore proteins regulated by HDHD5, LM2-4175 cells stably expressing an empty vector control (shNC) or shHDHD5#1 were analyzed by data-independent acquisition (DIA)–based quantitative proteomics, which was conducted by Westlake Omics (Westlake Omics, Hangzhou, China). Comparative analysis between the two groups was carried out to identify differentially expressed proteins using the t-test. Proteins with an absolute log₂fold change greater than 1 and P value below 0.05 were considered significantly differentially expressed.

Xenograft tumor models

Mycoplasma-free cells were orthotopically injected into the mammary fat pads of 5-week-old female BALB/c nude mice. Tumor growth was monitored by measuring tumor volume every two days after palpable tumors formed. Approximately 30 days after implantation, mice were euthanized and tumors were excised for further analysis. All animal experiments were conducted in accordance with institutional guidelines and were approved by the Institutional Animal Care and Use Committee of the Fudan University Shanghai Cancer Center (FUSCC).

Statistical analysis

All experiments were conducted with at least three independent replicates, and results are expressed as the mean ± standard deviation. Differences between two groups were analyzed using an unpaired, two-tailed Student’s t test, whereas comparisons among multiple groups were performed using one-way analysis of variance (ANOVA). Survival outcomes were evaluated using Kaplan–Meier analysis with the log-rank test. All statistical analyses were carried out using GraphPad Prism 9. A P value < 0.05 was considered statistically significant, with significance levels indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

Results

HDHD5 was highly expressed in TNBC and was associated with poor patient prognosis

To investigate the role of mitochondria-related genes in triple-negative breast cancer (TNBC), we analyzed transcriptomic and proteomic data from our previously established Fudan University Shanghai Cancer Center (FUSCC) TNBC dataset, together with transcriptomic data from the CBCGA dataset (Fig. 1A). We first performed differential expression analyses between normal and tumor samples using transcriptomic (screening criteria: log2FC > 0.6 and adjusted p < 0.05) and proteomic (screening criteria: log2FC > 2 and adjusted p < 0.05) data from the FUSCC-TNBC cohort. This analysis identified 44 mitochondria-related genes that were consistently upregulated in tumor cells at both the transcript and protein levels. We next sought to identify genes specifically overexpressed in TNBC. Using the CBCGA cohort, we performed differential expression analyses comparing TNBC with other breast cancer subtypes (screening criteria: log2 fold change > 0.5 and adjusted P < 0.05). This approach narrowed the candidates to 13 genes (Fig. 1B). As 12 of these genes had already been extensively studied in cancer, we selected HDHD5 for further investigation. In addition, we performed a pan-cancer analysis of HDHD5 expression using the TIMER2.0 dataset (http://timer.cistrome.org/). HDHD5 expression was significantly higher in tumor tissues than in normal tissues across most cancer types, including breast cancer (Fig. 1C). In the FUSCC TNBC dataset, HDHD5 mRNA and protein levels were significantly higher in TNBC tumor tissues than in normal tissues (Fig. 2A-B). Consistent with these findings, HDHD5 expression was also significantly elevated in TNBC tumors in the TCGA dataset (Fig. 2D). We next examined HDHD5 mRNA expression in normal breast tissue and across the three major molecular subtypes of breast cancer (HR+/HER2−, HER2+, and TNBC). In both the CBCGA (Fig. 2C) and TCGA (Fig. 2D) datasets, HDHD5 expression was highest in the TNBC subtype. To further validate these findings, we performed immunoblotting analyses on tumor samples and paired adjacent normal tissues from 15 patients with TNBC breast cancer. The results showed that HDHD5 expression was markedly increased in tumor specimens relative to adjacent normal tissues (Fig. 2E-F). Finally, we performed prognostic analyses using the Kaplan–Meier Plotter datasets. High HDHD5 (also known as CECR5; probe ID: 218592_s_at) expression was significantly associated with poor overall survival (OS), relapse-free survival (RFS), and distant metastasis-free survival (DMFS) in patients with TNBC (Fig. 2G-I, respectively). Overall, these results demonstrate that HDHD5 is highly expressed in TNBC and that high HDHD5 levels are associated with poor patient prognosis.

Fig. 1.

Fig. 1

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Identification of key mitochondria-related genes. (A) Venn diagram showing the overlap between differentially expressed genes identified from the FUSCC-TNBC transcriptomic and proteomic analyses, TNBC-specific upregulated genes from the CBCGA transcriptomic dataset, and the mitochondrial gene set from MitoCarta3.0. (B) Heatmap illustrating the expression patterns of 13 candidate mitochondria-related genes identified in the FUSCC-TNBC mRNA cohort (Normal: n = 88; TNBC: n = 360). (C) Pan-cancer analyses of HDHD5 expression status in normal and tumor tissues using the TIMER2.0 (http://timer.cistrome.org/) dataset; blue indicates normal tissue, red indicates tumor tissue, and purple indicates metastatic tissue. *, p < 0.05; **, p < 0.01; ***, p < 0.001

Fig. 2.

Fig. 2

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HDHD5 was highly expressed in TNBC and was associated with poor patient prognosis. (A-B) Analysis of HDHD5 mRNA (Normal: n = 88; TNBC: n = 360) (A) and protein (Normal: n = 26; TNBC: n = 76) (B) expression in primary TNBC tissues and paired adjacent normal tissues from the FUSCC-TNBC dataset. (C-D)Analysis of HDHD5 mRNA expression across different breast cancer subtypes in the CBCGA (Normal: n = 60; Luminal: n = 468; HER2: n = 181; TNBC: n = 103) (C) and TCGA (Normal: n = 113; Luminal: n = 639; HER2: n = 207; TNBC: n = 170) (D) datasets. (E-F） Western blot analysis of HDHD5 expression in 15 pairs of TNBC tissues and matched adjacent normal breast tissues (E), with the corresponding quantitative analysis shown in (F). (G-I) Association of HDHD5 expression with overall survival (OS) (high: n = 63; low: n = 138) (G), relapse-free survival (high: n = 235; low: n = 299) (RFS) (H), and distant metastasis-free survival (DMFS) (high: n = 118; low: n = 306) (I) in TNBC patients from the Kaplan–Meier Plotter database. *, p < 0.05; **, p < 0.01; ***, p < 0.001

HDHD5 promotes cell proliferation and colony formation in TNBC

To investigate the biological function of HDHD5 in TNBC, we conducted a series of functional assays. We first assessed HDHD5 protein expression by immunoblotting in eight TNBC cell lines and the normal breast epithelial cell line HMEC. HDHD5 expression was relatively high in LM2-4175 and SUM159 cell lines, whereas lower levels were observed in MDA-MB-231 and BT549 cell lines (Fig. 3A). Accordingly, we established stable HDHD5 knockdown in the LM2-4175 and SUM159 cell lines and stable HDHD5 overexpression in the MDA-MB-231 and BT549 cell lines (Fig. 3B-C). CCK-8 assays showed that HDHD5 knockdown significantly suppressed cell proliferation in the LM2-4175 and SUM159 cell lines (Fig. 3D-E). Consistently, colony formation assays demonstrated that silencing HDHD5 markedly reduced the clonogenic capacity of both LM2-4175 and SUM159 cells (Fig. 3F-G). In contrast, HDHD5 overexpression in the MDA-MB-231 and BT549 cell lines significantly enhanced cell proliferation and colony-forming capacity (Fig. 3H-K). Collectively, these results indicate that HDHD5 promotes cell proliferation and colony formation in TNBC.

Fig. 3.

Fig. 3

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HDHD5 promotes cell proliferation and colony formation in TNBC. (A) HDHD5 protein expression was assessed by immunoblotting in eight TNBC cell lines and a human mammary epithelial cell line. (B) Immunoblotting with the indicated antibodies was used to validate HDHD5 knockdown in LM2-4175 and SUM159 cells stably expressing shNC or shHDHD5. (C) Immunoblotting with the indicated antibodies was used to validate HDHD5 overexpression in MDA-MB-231 and BT549 cells stably expressing pLVX or HA-HDHD5. (D-G) LM2-4175 and SUM159 cells stably expressing shNC or shHDHD5 were subjected to CCK-8–based cell proliferation assays (D-E) and colony formation assays (F), with the corresponding quantitative analysis of colony formation shown in (G). (H-K) MDA-MB-231 and BT549 cells stably expressing pLVX or HA-HDHD5 were subjected to CCK-8–based cell proliferation assays (H-I) and colony formation assays (J), with the corresponding quantitative analysis of colony formation shown in (K). *, p < 0.05; **, p < 0.01; ***, p < 0.001

HDHD5 promotes cell migration and invasion in TNBC

High migratory and metastatic capacity is a key pathological feature of TNBC cells(Kwon et al. 2024, Błaszczak et al. 2025). We therefore evaluated the effects of HDHD5 on the migratory and invasive abilities of TNBC cells. Transwell assays were used to assess cell migration. Knockdown of HDHD5 significantly reduced migratory capacity in the LM2-4175 and SUM159 cell lines compared with control cells (Fig. 4A-B). In contrast, HDHD5 overexpression in the MDA-MB-231 and BT549 cell lines markedly enhanced cell migration (Fig. 4C-D). We next assessed the effects of HDHD5 on invasive capacity using transwell chambers pre-coated with Matrigel. HDHD5 knockdown significantly reduced the number of invaded cells in the LM2-4175 and SUM159 cell lines (Fig. 4E-F). Conversely, HDHD5 overexpression in the MDA-MB-231 and BT549 cell lines markedly increased the number of invading cells (Fig. 4G-H). Together, these findings suggest that HDHD5 enhances migration and invasion of TNBC cells.

Fig. 4.

Fig. 4

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HDHD5 promotes cell migration and invasion in TNBC. (A-B) LM2-4175 and SUM159 cells stably expressing shNC or shHDHD5 were subjected to Transwell assays to assess cell migration (A), and the corresponding quantitative results are shown in (B). Scale bar: 200 μm. (C-D) MDA-MB-231 and BT549 cells stably expressing pLVX or HA-HDHD5 were subjected to Transwell assays to assess cell migration (C), and the corresponding quantitative results are shown in (D). Scale bar: 200 μm. (E-F) LM2-4175 and SUM159 cells stably expressing shNC or shHDHD5 were subjected to Transwell assays to assess cell invasion (E), and the corresponding quantitative results are shown in (F). Scale bar: 200 μm. (G-H) MDA-MB-231 and BT549 cells stably expressing pLVX or HA-HDHD5 were subjected to Transwell assays to assess cell invasion (G), and the corresponding quantitative results are shown in (H). Scale bar: 200 μm. *, p < 0.05; **, p < 0.01; ***, p < 0.001

HDHD5 facilitates EMT-associated phenotypes and enhances migration and invasion in TNBC cell lines partly through S100A4

To investigate the downstream molecular mechanisms, we performed DIA-based quantitative proteomics in LM2-4175 cells with or without stable HDHD5 depletion (Fig. 5A). Differential expression analysis was conducted using a P value < 0.05 and an absolute log₂fold change > 1. This analysis identified 346 upregulated and 360 downregulated proteins, with HDHD5 among the significantly reduced (Fig. 5A-B). Further examination of the proteomic data revealed S100A4 as the most markedly downregulated protein, prompting subsequent validation by immunoblotting (Fig. 5C). The results showed that HDHD5 knockdown significantly reduced S100A4 protein levels in the LM2-4175 and SUM159 cell lines (Fig. 5D), whereas HDHD5 overexpression markedly increased S100A4 protein expression in the MDA-MB-231 and BT549 cell lines (Fig. 5E). To further clarify how HDHD5 regulates S100A4, we performed RT-qPCR analysis. In LM2-4175 and SUM159 cells, HDHD5 knockdown significantly reduced S100A4 mRNA expression (Fig. S1A). Conversely, HDHD5 overexpression in MDA-MB-231 and BT549 cells significantly increased S100A4 mRNA levels (Fig. S1B). Together, these findings suggest that HDHD5 regulates S100A4 expression at the transcriptional level.

Fig. 5.

Fig. 5

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HDHD5 facilitates EMT-associated phenotypes and enhances migration and invasion in TNBC cell lines partly through S100A4. (A) Volcano plot showing differentially expressed proteins identified by data-independent acquisition (DIA)–based quantitative proteomics between LM2-4175 cells stably expressing shNC and shHDHD5#1. (B-C) Bar plots showing the expression levels of HDHD5 and S100A4 between the shNC and shHDHD5#1 groups based on quantitative proteomic analysis. (D) Immunoblotting was used to detect the levels of S100A4 in LM2-4175 and SUM159 cells stably expressing shNC or shHDHD5#1 or shHDHD5#2. (E) Immunoblotting was used to detect the levels of S100A4 in MDA-MB-231 and BT549 cells stably expressing pLVX or HA-HDHD5. (F) LM2-4175 and SUM159 cells stably expressing shNC or shHDHD5#1, alone or in combination with Flag-S100A4, were subjected to immunoblotting to assess the expression of E-cadherin, N-cadherin, and vimentin. (G-H) LM2-4175 and SUM159 cells stably expressing shNC or shHDHD5#1, alone or in combination with Flag-S100A4, were subjected to transwell assays to assess cell migration (G), and the corresponding quantitative results are shown in (H). Scale bar: 200 μm. (I-J) LM2-4175 and SUM159 cells stably expressing shNC or shHDHD5#1, alone or in combination with Flag-S100A4, were subjected to transwell assays to assess cell invasion (I), and the corresponding quantitative results are shown in (J). Scale bar: 200 μm. *, p < 0.05; **, p < 0.01; ***, p < 0.001

We next performed rescue experiments. S100A4 was introduced into LM2-4175 and SUM159 cell lines following HDHD5 knockdown, and successful expression was confirmed by immunoblotting (Fig. 5F). Previous studies have shown that S100A4 is involved in the EMT process in tumors(Chow et al. 2017, Low et al. 2023, Deng et al. 2024, Zhang et al. 2023). Therefore, we examined the expression levels of EMT markers, including E-cadherin, N-cadherin, and vimentin. Consistently, HDHD5 depletion led to a marked increase in the epithelial marker E-cadherin and a concomitant decrease in the mesenchymal markers N-cadherin and vimentin in both cell lines. Re-expression of S100A4 effectively reversed the changes in E-cadherin, N-cadherin, and vimentin induced by HDHD5 knockdown (Fig. 5F). Finally, transwell assays were performed using the cell lines shown in Fig. 5F. The results showed that restoration of S100A4 expression partly reversed the reduced migratory and invasive abilities caused by HDHD5 knockdown in LM2-4175 and SUM159 cells (Fig. 5G-J).

Overall, HDHD5 can promote the EMT-associated phenotypes and enhance migratory and invasive capacities by regulating S100A4 expression.

HDHD5 promotes TNBC growth in vitro and in vivo partly through S100A4

We next investigated whether the pro-proliferative effect of HDHD5 in TNBC is mediated by S100A4. CCK-8 assays in LM2-4175 and SUM159 cells showed that restoration of S100A4 partially rescued the proliferation deficit induced by HDHD5 knockdown (Fig. 6A-B). Colony formation assays further showed that overexpression of S100A4 following HDHD5 knockdown significantly restored the clonogenic capacity of LM2-4175 and SUM159 cells (Fig. 6C-D).

Fig. 6.

Fig. 6

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HDHD5 promotes TNBC growth in vitro and in vivo partly through S100A4. (A-D) LM2-4175 and SUM159 cells stably expressing shNC or shHDHD5#1, alone or in combination with Flag-S100A4, were subjected to CCK-8 assays(A-B) and colony formation assays (C), with the corresponding quantitative analysis of colony formation shown in (D). (E-G) Orthotopic xenograft models were established in female BALB/c nude mice (n = 6 per group) using LM2-4175 cells expressing shNC or shHDHD5#1, with or without Flag-S100A4. Representative images of excised tumors (E), together with tumor weight (F) and volume (G) measurements, are shown. *, p < 0.05; **, p < 0.01; ***, p < 0.001

To further validate the in vitro findings, LM2-4175 cells stably expressing shNC, shHDHD5, or shHDHD5 combined with S100A4 overexpression were orthotopically injected into 5-week-old female BALB/c nude mice. As shown in Fig. 6E-G, tumors derived from the shHDHD5 group grew significantly more slowly than those from the shNC group (Fig. 6E). Consistently, both tumor weight and tumor volume were markedly reduced in the shHDHD5 group compared with the shNC group (Fig. 6F-G). Importantly, reintroduction of S100A4 into LM2-4175 cells stably expressing shHDHD5 partially rescued tumor growth capacity, as evidenced by increased tumor growth rate, tumor volume, and tumor weight compared with the shHDHD5 group alone (Fig. 6F-G).

Overall, these results indicate that HDHD5 promotes the tumorigenic capacity of TNBC in vitro and in vivo, at least in part, through S100A4.

Discussion

Triple-negative breast cancer (TNBC) is a biologically and clinically heterogeneous disease that remains a major unmet clinical challenge due to its aggressive behavior, poor prognosis, and continued reliance on chemotherapy as the primary treatment option (Bianchini et al. 2016). This heterogeneity is driven by diverse genetic alterations and aberrant activation of multiple signaling pathways, which has enabled the development of targeted strategies, including therapies directed at DNA repair pathways, androgen receptor signaling, and specific kinases (Li et al. 2022). To move beyond empirical treatment approaches, a deeper understanding of the molecular features is essential for guiding more precise and effective therapeutic interventions.

In this study, multi-omics analyses identified the mitochondrial-associated gene HDHD5, which has not been previously investigated in cancer, as being specifically overexpressed in TNBC. HDHD5 promotes TNBC growth and progression and is associated with poor patient outcomes. Mechanistically, HDHD5 promotes EMT-associated phenotypes and tumor growth by regulating S100A4 expression.

First, we report for the first time that HDHD5 plays a pro-tumorigenic role in TNBC. In previous studies, HDHD5 has been indicated as a mitochondrial-associated gene involved in lipid regulation (Jiang et al. 2022).More recently, HDHD5 has been identified as part of a mitochondrial lipid metabolism–related gene signature with prognostic value in colorectal cancer (Wang et al. 2025). However, the functional and mechanistic roles of HDHD5 in human cancers remain unexplored. Through integrative multi-omics analyses of FUSCC TNBC, CBCGA and TCGA datasets, together with validation by immunoblotting in clinical samples, we confirmed that HDHD5 is specifically upregulated in TNBC, and that its elevated expression is associated with poor prognosis. However, despite this strong prognostic relevance, HDHD5 levels lacked statistically significant correlations with specific clinicopathological features, highlighting the need for larger cohorts to fully elucidate its clinical significance. Nevertheless, our analysis of the TIMER2.0 dataset shows that HDHD5 is overexpressed across multiple cancer types, implying broader relevance beyond TNBC. This observation raises the possibility that HDHD5 may play a similar oncogenic role in other malignancies, which merits further investigation.

Second, sustaining proliferative signaling is one of the hallmarks of cancer (Hanahan 2022). Therefore, we examined the pro-proliferative effects of HDHD5 and found that HDHD5 significantly promotes TNBC cell proliferation both in vivo and in vitro. In addition, TNBC exhibits a highly aggressive nature, and clinical evidence suggests that distant metastasis is the primary cause of death in most patients (Zardavas et al. 2015). In our study, knockdown of HDHD5 markedly suppressed the migratory and invasive capacities of TNBC cells, further supporting an important role for HDHD5 in the aggressive nature of TNBC. Overall, HDHD5 promotes malignant behaviors in TNBC.

Third, we found that HDHD5 exerts its tumor-promoting functions by upregulating the oncogenic factor S100A4. Our quantitative proteomic analysis revealed that S100A4 was the most significantly downregulated protein following HDHD5 knockdown. We confirmed this finding at the protein level using immunoblotting. In addition, RT-qPCR experiments further demonstrated that HDHD5 regulates S100A4 expression by increasing its mRNA levels, suggesting a direct or indirect transcriptional regulatory mechanism. The pro-tumorigenic roles of S100A4 in cancer have been widely reported, including regulation of EMT process (Low et al. 2023), promotion of tumor metastasis (Sun et al. 2021), contribution to therapeutic resistance (Yan et al. 2025), and modulation of tumor immunity(Abdelfattah et al. 2022). Hu H et al. found that targeting lysosomal degradation of S100A4 regulates the proliferation and metastasis of triple-negative breast cancer (Hu et al. 2025). Park WY et al. reported that S100A4 mediates metastatic outgrowth in breast cancer cells(Park et al. 2023). A previous study has showed that elevated S100A4 enhances the expression of metastasis-associated effector molecules in human breast cancer (Ismail et al. 2017). Moreover, EGFR–ERK–S100A4 signaling has been implicated in promoting breast cancer lung metastasis (Liu et al. 2023). Consistent with these findings, our rescue experiments demonstrated that the inhibitory effects of HDHD5 knockdown on TNBC cell proliferation, migration, and invasion were mediated by S100A4. These results provide new insight into the upstream regulatory mechanisms of S100A4 in breast cancer. However, although our functional rescue experiments establish S100A4 as a critical downstream mediator of HDHD5-induced EMT-associated phenotypes and growth, it is important to note that S100A4 is unlikely to be the exclusive effector. Our proteomic analysis revealed a broad spectrum of differentially expressed proteins following HDHD5 alteration. This indicates that HDHD5 likely operates through a more comprehensive regulatory network in TNBC. S100A4 serves as a key driver within this network, but the contribution of other identified downstream targets to TNBC progression warrants further investigation in our future studies.

Beyond this intricate downstream signaling network, as a mitochondrial-associated protein, HDHD5 provides a unique perspective on the interplay between mitochondrial function, metabolic reprogramming, and tumor metastasis. The execution of EMT-associated phenotypes is a highly energy-consuming process that heavily relies on metabolic shifts, with mitochondrial metabolism playing a pivotal role (Sciacovelli and Frezza 2017, Porporato et al. 2014). HDHD5 may contribute to TNBC progression by modulating mitochondrial metabolic pathways, thereby creating a favorable metabolic microenvironment that supports S100A4-driven invasive behaviors. Future studies will focus on delineating the specific metabolic reprogramming events induced by HDHD5 in TNBC, with the aim of targeting mitochondrial vulnerabilities to overcome current therapeutic challenges.

In summary, HDHD5 is specifically overexpressed in TNBC and is associated with poor prognosis. HDHD5 promotes TNBC cell migration and invasion in vitro and enhances TNBC cell proliferation both in vitro and in vivo. Mechanistically, HDHD5 facilitates TNBC proliferation and EMT-associated phenotypes by upregulating S100A4 expression. Our study identifies HDHD5 as a previously unrecognized driver of tumor progression in TNBC and supports its potential value as a therapeutic target.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (111.4KB, docx)

Acknowledgements

We would like to express our gratitude to the clinical and basic research teams of the Department of Breast Surgery and Shanghai Key Laboratory of Breast cancer at Fudan University Shanghai Cancer Center for their technical and resource support in this study.

Author contributions

JYC: Data curation, formal analysis, investigation, and writing the manuscript draft. YLZ, FLZ, QZ and MKM: Data analysis and investigation. ZMS: Methodology, supervision, funding acquisition, project administration, and manuscript editing.

Funding

Not applicable.

Data availability

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

Declarations

Conflict of interest

The authors declare no potential conflicts of interest.

Footnotes

Publisher’s note

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Contributor Information

Jia-Yang Cai, Email: jiaycai@whu.edu.cn.

Zhi-Min Shao, Email: zhimingshao@fudan.edu.cn.

References

  1. Bianchini G, De Angelis C, Licata L, Gianni L (2022) Treatment landscape of triple-negative breast cancer - expanded options, evolving needs. Nat Rev Clin Oncol 19(2):91–113 [DOI] [PubMed] [Google Scholar]
  2. Bianchini G, Balko JM, Mayer IA, Sanders ME, Gianni L (2016) Triple-negative breast cancer: challenges and opportunities of a heterogeneous disease. Nat Rev Clin Oncol 13(11):674–690 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Li Y, Zhang H, Merkher Y, Chen L, Liu N, Leonov S et al (2022) Recent advances in therapeutic strategies for triple-negative breast cancer. J Hematol Oncol 15(1):121 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Nunnari J, Suomalainen A (2012) Mitochondria: in sickness and in health. Cell 148(6):1145–1159 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Tanprasert P, Limpakan Yamada S, Chattipakorn SC, Chattipakorn N, Shinlapawittayatorn K (2022) Targeting mitochondria as a therapeutic anti-gastric cancer approach. Apoptosis 27(3–4):163–183 [DOI] [PubMed] [Google Scholar]
  6. Xiong Z, Yang L, Zhang C, Huang W, Zhong W, Yi J et al (2025) MANF facilitates breast cancer cell survival under glucose-starvation conditions via PRKN-mediated mitophagy regulation. Autophagy 21(1):80–101 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Xu T, Chu C, Xue S, Jiang T, Wang Y, Xia W et al (2025) Identification and validation of a prognostic signature of drug resistance and mitochondrial energy metabolism-related differentially expressed genes for breast cancer. J Transl Med 23(1):131 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ding P, Pei S, Qu Z, Yang Y, Liu Q, Kong X et al (2024) Single-cell sequencing unveils mitophagy-related prognostic model for triple-negative breast cancer. Front Immunol 15:1489444 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Yu S, Zhou Y, Zhang S, He F, Jiang L, Lv X et al (2025) The role and prognostic value of mitochondria-related gene PARK7 in breast cancer. Breast Cancer Res 27(1):200 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jiang YZ, Ma D, Suo C, Shi J, Xue M, Hu X et al (2019) Genomic and Transcriptomic Landscape of Triple-Negative Breast Cancers: Subtypes and Treatment Strategies. Cancer Cell 35(3):428–40e5 [DOI] [PubMed] [Google Scholar]
  11. Jiang YZ, Ma D, Jin X, Xiao Y, Yu Y, Shi J et al (2024) Integrated multiomic profiling of breast cancer in the Chinese population reveals patient stratification and therapeutic vulnerabilities. Nat Cancer 5(4):673–690 [DOI] [PubMed] [Google Scholar]
  12. Jiang X, Xu Z, Yao D, Liu X, Liu W, Wang N et al (2022) An integrated multi-omics approach revealed the regulation of melatonin on age-dependent mitochondrial function impair and lipid dyshomeostasis in mice hippocampus. Pharmacol Res 179:106210 [DOI] [PubMed] [Google Scholar]
  13. Wang H, Zhang K, Wang Y, Chen M, Zhang M (2025) A mitochondrial lipid metabolism-related gene signature predicts prognosis and immune landscape in colorectal cancer. Front Immunol 16:1669678 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Rath S, Sharma R, Gupta R, Ast T, Chan C, Durham TJ et al (2021) MitoCarta3.0: an updated mitochondrial proteome now with sub-organelle localization and pathway annotations. Nucleic Acids Res 49(D1):D1541–d7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Li T, Fu J, Zeng Z, Cohen D, Li J, Chen Q et al (2020) TIMER2.0 for analysis of tumor-infiltrating immune cells. Nucleic Acids Res 48(W1):W509–w14 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Győrffy B (2021) Survival analysis across the entire transcriptome identifies biomarkers with the highest prognostic power in breast cancer. Comput Struct Biotechnol J 19:4101–4109 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kwon MR, Park JS, Ko EJ, Park J, Ju EJ, Shin SH et al (2024) Ibulocydine Inhibits Migration and Invasion of TNBC Cells via MMP-9 Regulation. Int J Mol Sci. ;25(11) [DOI] [PMC free article] [PubMed]
  18. Błaszczak E, Miziak P, Odrzywolski A, Baran M, Gumbarewicz E, Stepulak A (2025) Triple-Negative Breast Cancer Progression and Drug Resistance in the Context of Epithelial-Mesenchymal Transition. Cancers (Basel). ;17(2) [DOI] [PMC free article] [PubMed]
  19. Chow KH, Park HJ, George J, Yamamoto K, Gallup AD, Graber JH et al (2017) S100A4 Is a Biomarker and Regulator of Glioma Stem Cells That Is Critical for Mesenchymal Transition in Glioblastoma. Cancer Res 77(19):5360–5373 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Low RRJ, Fung KY, Gao H, Preaudet A, Dagley LF, Yousef J et al (2023) S100 family proteins are linked to organoid morphology and EMT in pancreatic cancer. Cell Death Differ 30(5):1155–1165 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Deng D, Begum H, Liu T, Zhang J, Zhang Q, Chu TY et al (2024) NFAT5 governs cellular plasticity-driven resistance to KRAS-targeted therapy in pancreatic cancer. J Exp Med. ;221(11) [DOI] [PMC free article] [PubMed]
  22. Zhang M, Barroso E, Ruart M, Peña L, Peyman M, Aguilar-Recarte D et al (2023) Elafibranor upregulates the EMT-inducer S100A4 via PPARβ/δ. Biomed Pharmacother 167:115623 [DOI] [PubMed] [Google Scholar]
  23. Hanahan D (2022) Hallmarks of Cancer: New Dimensions. Cancer Discov 12(1):31–46 [DOI] [PubMed] [Google Scholar]
  24. Zardavas D, Irrthum A, Swanton C, Piccart M (2015) Clinical management of breast cancer heterogeneity. Nat Rev Clin Oncol 12(7):381–394 [DOI] [PubMed] [Google Scholar]
  25. Sun H, Wang C, Hu B, Gao X, Zou T, Luo Q et al (2021) Exosomal S100A4 derived from highly metastatic hepatocellular carcinoma cells promotes metastasis by activating STAT3. Signal Transduct Target Ther 6(1):187 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Yan YY, Deng ZF, Wu XT, Lu Y, Zhu ZY, Wen Q et al (2025) Low miR-224-5p in exosomes confers colorectal cancer 5-FU resistance by upregulating S100A4. Drug Resist Updat 79:101211 [DOI] [PubMed] [Google Scholar]
  27. Abdelfattah N, Kumar P, Wang C, Leu JS, Flynn WF, Gao R et al (2022) Single-cell analysis of human glioma and immune cells identifies S100A4 as an immunotherapy target. Nat Commun 13(1):767 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hu H, Bai H, Wang C, Xi L, Tian S, Ni M et al (2025) Super enhancer lncRNA RP11-54O7.17 regulates the proliferation and metastasis of triple-negative breast cancer by targeting lysosomal degradation of S100A4. Cell Death Dis 16(1):773 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Park WY, Gray JM, Holewinski RJ, Andresson T, So JY, Carmona-Rivera C et al (2023) Apoptosis-induced nuclear expulsion in tumor cells drives S100a4-mediated metastatic outgrowth through the RAGE pathway. Nat Cancer 4(3):419–435 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ismail TM, Bennett D, Platt-Higgins AM, Al-Medhity M, Barraclough R, Rudland PS (2017) S100A4 Elevation Empowers Expression of Metastasis Effector Molecules in Human Breast Cancer. Cancer Res 77(3):780–789 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Liu A, Li Y, Lu S, Cai C, Zou F, Meng X (2023) Stanniocalcin 1 promotes lung metastasis of breast cancer by enhancing EGFR-ERK-S100A4 signaling. Cell Death Dis 14(7):395 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sciacovelli M, Frezza C (2017) Metabolic reprogramming and epithelial-to-mesenchymal transition in cancer. Febs j 284(19):3132–3144 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Porporato PE, Payen VL, Pérez-Escuredo J, De Saedeleer CJ, Danhier P, Copetti T et al (2014) A mitochondrial switch promotes tumor metastasis. Cell Rep 8(3):754–766 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (111.4KB, docx)

Data Availability Statement

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

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