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Liuetal. Cell Communication and Signaling (2025) 23:22
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Cell Communication
and Signaling
Targeting theERK1/2 andp38 MAPK
pathways attenuates Golgi tethering factor
golgin-97 depletion-induced cancer progression
inbreast cancer
Yu-Chin Liu1, Tsung-Jen Lin1,2, Kowit-Yu Chong3,4,5,6, Guan-Ying Chen1, Chia-Yu Kuo1, Yi-Yun Lin7,
Chia-Wei Chang7, Ting-Feng Hsiao7, Chih-Liang Wang8,9, Yo-Chen Shih7 and Chia-Jung Yu1,7,9,10*
Abstract
Background The Golgi apparatus is widely considered a secretory center and a hub for different signaling pathways.
Abnormalities in Golgi dynamics can perturb the tumor microenvironment and influence cell migration. Therefore,
unraveling the regulatory network of the Golgi and searching for pharmacological targets would facilitate the devel-
opment of novel anticancer therapies. Previously, we reported an unconventional role for the Golgi tethering factor
golgin-97 in inhibiting breast cell motility, and its downregulation was associated with poor patient prognosis. How-
ever, the specific role and regulatory mechanism of golgin-97 in cancer progression in vivo remain unclear.
Methods We integrated genetic knockout (KO) of golgin-97, animal models (zebrafish and xenograft mice), multi-
omics analysis (next-generation sequencing and proteomics), bioinformatics analysis, and kinase inhibitor treatment
to evaluate the effects of golgin-97 KO in triple-negative breast cancer cells. Gene knockdown and kinase inhibitor
treatment followed by qRT‒PCR, Western blotting, cell viability, migration, and cytotoxicity assays were performed
to elucidate the mechanisms of golgin-97 KO-mediated cancer invasion. A xenograft mouse model was used to inves-
tigate cancer progression and drug therapy.
Results We demonstrated that golgin-97 KO promoted breast cell metastasis in zebrafish and xenograft mouse mod-
els. Multi-omics analysis revealed that the Wnt signaling pathway, MAPK kinase cascades, and inflammatory cytokines
are involved in golgin-97 KO-induced breast cancer progression. Targeting the ERK1/2 and p38 MAPK pathways
effectively attenuated golgin-97-induced cancer cell migration, reduced the expression of inflammatory mediators,
and enhanced the chemotherapeutic effect of paclitaxel in vitro and in vivo. Specifically, compared with the paclitaxel
regimen, the combination of ERK1/2 and p38 MAPK inhibitors significantly prevented lung metastasis and lung injury.
We further demonstrated that hypoxia is a physiological condition that reduces golgin-97 expression in cancer, reveal-
ing a novel and potential feedback loop between ERK/MAPK signaling and golgin-97.
Conclusion Our results collectively support a novel regulatory role of golgin-97 in ERK/MAPK signaling
and the tumor microenvironment, possibly providing new insights for anti-breast cancer drug development.
Keywords Golgi, Golgin-97, Breast cancer, MAPK signaling, Metastasis, Inflammatory cytokine
*Correspondence:
Chia-Jung Yu
yucj1124@mail.cgu.edu.tw; yucj1124@gmail.com
Full list of author information is available at the end of the article
Page 2 of 18
Liuetal. Cell Communication and Signaling (2025) 23:22
Introduction
Breast cancer is the most common type of cancer in
female patients and the second leading cause of death
among women overall [1]. Triple-negative breast can-
cer (TNBC) has been shown to have a higher incidence
of metastasis, a poorer prognosis, and poorer overall
survival than other subtypes of breast cancer [2]. TNBC
lacks the expression of the sensitive therapeutic mark-
ers estrogen receptor, progesterone receptor, and human
epidermal growth factor receptor 2, leading to the inef-
fectiveness of endocrine therapy. Systemic chemotherapy
with anthracyclines and taxanes is the most common
first-line treatment modality [3]. However, anticancer
drug resistance, off-target toxicity, and complications,
including pneumonia with severe inflammation, are
major obstacles to the development of cancer chemo-
therapy regimens [4–6]. Although the possible molecu-
lar mechanisms of TNBC tumor growth, metastasis, and
therapy have been increasingly revealed, their clinical
application is still limited. us, identifying new molec-
ular targets for predicting clinical prognosis and finding
effective strategies for treating TNBC are crucial.
e Golgi apparatus is the trafficking hub that regu-
lates the anterograde transport of modified proteins and
lipids or retrieves extracellular proteins through retro-
grade transport from endosomes [7]. ere is growing
evidence that the Golgi apparatus is a hub for different
signaling pathways [8–10]. Several Golgi-localized pro-
teins have recently been reported to be involved in the
function of the Golgi and are related to the regulation of
cancer cell migration and invasion [10, 11]. Golgin-97,
localized at the trans-Golgi network (TGN), is a coiled-
coil protein first identified in Sjogren’s syndrome patients
[12]. Golgin-97 maintains Golgi integrity and acts as a
tethering molecule that mediates the vesicular traffick-
ing of specific cargoes (such as E-cadherin) [13–15]. Gol-
gin-97 expression is significantly lower in invasive ductal
breast cancer than in ductal carcinoma insitu [16]. Low
expression of golgin-97 has also been positively associ-
ated with poor overall survival in patients with breast,
lung, and ovarian cancers, supporting a suppressive
role of golgin-97 in regulating cancer progression. We
then revealed a noncanonical role of golgin-97 in sup-
pressing TNBC cell motility through the modulation of
NF-κB activity in vitro [17]. Unfortunately, the inhibi-
tion of NF-κB alone is insufficient to induce pronounced
apoptosis in tumor cells, and long-term treatment with
NF-κB inhibitors influences normal cell function, lead-
ing to severe systemic toxicity and immunosuppression
[18]. Additionally, whether and how golgin-97 depletion
promotes cancer metastasis invivo and the mechanisms
affecting golgin-97 downregulation in breast cancer
remain unclear.
In this study, we established a cell line with stable
golgin-97 depletion via CRISPR‒Cas9 gene knock-
out (KO) and demonstrated that golgin-97 depletion
promoted MDA-MB-231 cell migration and metasta-
sis. Multi-omics analysis and functional characteriza-
tion demonstrated the therapeutic potential of p38
MAPK inhibitors and ERK1/2 inhibitors in golgin-97
KO-induced cancer progression in vitro and in vivo,
confirming their biological importance in breast cancer
and anticancer drug development.
Materials andmethods
Cell culture, chemical reagents, andchemotherapy drugs
e TNBC cell line MDA-MB-231 was purchased from
the American Type Culture Collection (Manassas, Vir-
ginia). Golgin-97 KO MDA-MB-231 cells were gener-
ated via CRISPR‒Cas9 gene KO in our laboratory. Both
wild-type MDA-MB-231 (hereafter referred to as MDA-
MB-231) cells and golgin-97 KO MDA-MB-231 cells
were cultured in Dulbecco’s modified Eagle’s medium
(DMEM) (Gibco, Waltham, MA, USA) containing 10%
FBS (Gibco), 1% penicillin/streptomycin (Gibco), and 1%
L-glutamine (Gibco). SB203580, U0126, and paclitaxel
were purchased from MedChemExpress (Monmouth
Junction, NJ, USA). Cobalt chloride (CoCl2) was pur-
chased from Sigma (St. Louis, MO, USA).
CRISPR‒Cas9 gene deletion
Golgin-97−/− KO MDA-MB-231 breast cancer cells were
generated with an optimized single-guide RNA (sgRNA;
human golgin-97, accession no. NM_002077.3) and the
Cas9 expression plasmid pRGEN-Cas9-CMV (ToolGen,
Seoul, Korea). Details for establishing cell lines with
golgin-97 KO are described in Appendix S1 (available
online).
Cancer metastasis inthezebrash model
e zebrafish xenotransplantation procedure was
described previously [19]. Briefly, 24 h postfertilization
(hpf), zebrafish embryos of the wild-type Tübingen strain
were dechorionated and anesthetized in E3 medium
containing phenylthiourea and tricaine before cell injec-
tion. MDA-MB-231 cells (control or golgin-97 KO) were
labeled with the lipophilic fluorochrome CM-DiI (Invit-
rogen, Waltham, MA, USA). Stained cells were injected
into the yolk sacs of zebrafish embryos via a microinjec-
tor according to the instruction manual. e injected
zebrafish were maintained in E3 medium. Images of the
zebrafish were acquired with a Z16 APO microscope
using a Leica DFC490 color camera system.
Page 3 of 18
Liuetal. Cell Communication and Signaling (2025) 23:22
Next‑generation sequencing (NGS) andbioinformatic
analysis
Total RNA was extracted from MDA-MB-231 and
golgin-97 KO cells via TRIzol Reagent (Invitrogen,
Waltham, MA, USA) according to the instruction man-
ual. NGS was carried out via Illumina’s sequencing-
by-synthesis technology (Illumina, USS) as described
previously [20]. For pathway enrichment analysis, the
differentially expressed genes (DEGs) whose expression
was altered twofold in golgin-97 KO cells compared
with that in MDA-MB-231 cells were subjected to path-
way analysis via DAVID Bioinformatics Resources 6.7
(https:// david-d. ncifc rf. gov/ home. jsp) and QIAGEN
Ingenuity Pathway Analysis (IPA) software.
Quantitative proteomic analysis ofthedierential
proteome resulting fromgolgin‑97 KO
Protein extracts prepared from MDA-MB-231 and gol-
gin-97 KO cells were digested with trypsin, chemically
labeled with a tandem mass tag (TMT), and then sub-
jected to two-dimensional liquid chromatography‒tan-
dem mass spectrometry (2D LC‒MS/MS). e details
for protein identification and quantification via mass
spectrometry are provided in Appendix S1 (available
online).
Small interfering RNA (siRNA) transfection
siRNAs targeting p38 MAPK, ERK1/2 and golgin-97
were purchased from ermo Fisher Scientific or
Dharmacon and used for gene knockdown with Lipo-
fectamine RNAiMAX reagents (Invitrogen, Grand
Island, NY, USA) according to the manufacturer’s
instructions. e sequences of the siRNAs used in this
study are listed in TableS1.
Cell viability assay
Cell viability was determined by using a Cell Counting
Kit-8 (CCK-8) assay (TEN-CCK8) (BIOTOOLS Co.,
Ltd., New Taipei City, Taiwan). e detailed methods
of the cell viability assay are available in Appendix S1
(available online).
Cell andanimal tissue RNA extraction andquantitative
RT‒PCR
Total RNA was extracted from cells or tumor tissue,
and cDNA was prepared for qRT‒PCR as described
previously [17]. e sequences of the primers used in
this study are listed in TableS2.
Western blotting
e detailed Western blotting methods are available
in Appendix S1. e primary antibodies used included
anti-golgin-97 (13192S), anti-IκBα (4814S), anti-p38
(8690S), anti-phospho-ERK1/2 (9101S), anti-cleaved
caspase-3 (#9661) and anti-caspase-3 (#9662), which
were purchased from Cell Signaling Technology (Dan-
vers, MA, USA). e anti-ERK (sc-93) antibody was
purchased from Santa Cruz (DT, USA). Anti-phospho-
p38 (09–272) and anti-β-actin (MAB1501) antibod-
ies were obtained from Merck Millipore (Burlington,
MA, USA). e anti-HIF1α (GTX127309) antibody was
purchased from GeneTex (Irvine, CA, USA). e anti-
TGN46 antibody was purchased from Bio-Rad (Hercu-
les, CA, USA).
Wound healing migration assay
e cell migration ability was analyzed via a wound heal-
ing assay. e detailed methods are available in Appendix
S1 (available online).
Animal model ofmetastasis
is study was approved by the Institutional Animal
Care and Use Committee of Chang Gung University
(CGU107–228). Four-week-old female immunodeficient
nonobese diabetic/SCID (NOD. CB17-Prkdcscid/JNarl)
mice were purchased from the National Laboratory
Animal Center (Taipei, Taiwan) to establish the tail vein
injection and orthotopic inoculation models as described
in Appendix S1 (available online).
Immunohistochemical (IHC) analysis
e detailed IHC methods are available in Appendix S1.
Statistical analysis
All experiments were performed at least in triplicate, and
the data are presented as the means ± standard devia-
tions (SDs). Prism 8.0 (GraphPad Software) statistical
software packages were used for data analysis. Unpaired t
tests, one-way ANOVA, two-way ANOVA, and log-rank
tests were used for comparisons between the control and
experimental groups. A P value less than 0.05 (P < 0.05)
was considered to indicate significance.
Results
Golgin‑97 KO modulates themalignancy ofbreast cancer
cells in vivo
Our previous studies revealed that golgin-97 knock-
down promotes NF-κB activation and leads to increased
migration/invasion of breast cancer cells [17]. Herein, we
applied CRISPR–Cas9 to generate four clonal golgin-97
KO cell lines (Fig. S1A). We confirmed that the expres-
sion levels of IκBα and TGN46 were reduced, accom-
panied by increased migration and invasion of 2–9 and
2–20 KO cells (Fig. S1B). erefore, to verify the effects
of golgin-97 depletion on cancer metastasis in vivo,
Page 4 of 18
Liuetal. Cell Communication and Signaling (2025) 23:22
cancer cells were injected into the perivitelline cavity of
fertilized zebrafish embryos. Four experiments were con-
ducted with ten fish per group, resulting in a total of more
than 30 zebrafish per group. e results indicated that
20% of the MDA-MB-231 cells had metastasized by day
one, an increase of 24% according to subsequent observa-
tions. In comparison, golgin-97 KO (2–9)cells presented
a greater metastasis rate, beginning at 46% on day one,
peaking at 52% on day two, and subsequently declining
to 36% on day three. Golgin-97 KO (2–20) cells pre-
sented lower rates of metastasis, with rates of 17%, 30%,
and 34%, respectively. In the present study, we present
the quantitative results obtained on day two, as shown in
Fig.1B. CM-DiI staining further revealed that the num-
ber of cancer cell clusters spread distal to the primary site
was significantly greater in fish injected with golgin-97
KO cells than in those injected with MDA-MB-231 cells
(Fig.1A and B). Furthermore, when MDA-MB-231 and
Fig. 1 Golgin-97 KO modulates the malignancy of breast cancer cells in vivo. A Representative images showing that MDA-MB-231 and golgin-97
KO cells were injected into the perivitelline cavity of zebrafish embryos and analyzed with a dissecting microscope. The location of the cancer cells
is indicated in red on bright field images of zebrafish. The white arrows indicate dispersed breast cancer cells. B Quantification of the number of cell
clusters on the basis of the acquired images in Fig. 1A. Each dot represents one live fish for image analysis. The quantitative data were analyzed
via one-way ANOVA. C Representative images of lung metastases formed from MDA-MB-231 and 2–9 KO cell-derived NOD/SCID mice on day
28 after tail vein injection. D Representative IHC images with H&E staining of lung metastases. Scale bars, 100 μm. The yellow arrows delineate
the tumor region. E Representative images showing the tumor sizes on day 42 after the injection of MDA-MB-231 and 2–9 KO cells into the right
middle mammary fat pads of the mice. F Tumor growth was monitored at the indicated times after the injection of MDA-MB-231 and 2–9 KO cells,
and tumor growth curves were constructed. The data were analyzed via two-way ANOVA. G Compared with those injected with MDA-MB-231
cells, NOD/SCID mice injected with golgin-97 KO cells exhibited poor survival (n = 9 mice per group). Kaplan–Meier plots of mouse survival
using a standard tumor volume of 300 mm3 as a criterion for euthanasia. P values were determined via the log-rank test. The data are presented
as the means ± SDs (*p < 0.05; **p < 0.01; ****, P < 0.0001)
Page 5 of 18
Liuetal. Cell Communication and Signaling (2025) 23:22
golgin-97 KO cells were injected into the lateral tail veins
of immunodeficient NOD/SCID mice, a trend toward the
formation of lung foci was observed in golgin-97 KO cell-
injected mice, suggesting that golgin-97 KO breast can-
cer cells have a greater rate of survival and colonization
success (Fig.1C). H&E staining revealed that golgin-97
KO increased the tumor cell density and resulted in the
formation of large nodules in the stroma (Fig.1D). us,
these differences in cell morphology and organization
suggest that golgin-97 KO enhances the invasive poten-
tial of cancer. In addition, the orthotopic mouse model
demonstrated that the tumors derived from golgin-97
KO cells were significantly larger than the tumors derived
from MDA-MB-231 cells (Fig.1E). Quantitative analysis
revealed a significant increase in tumor size in golgin-97
KO cell-injected mice on day 35 (Fig.1F). As expected,
the survival time of golgin-97 KO mice was shorter than
that of MDA-MB-231 mice (Fig.1G). ese results col-
lectively support the suppressive role of golgin-97 in the
tumor growth and metastatic progression of breast can-
cer invivo.
Multi‑omics reveals theinvolvement ofkey factors
ingolgin‑97 KO‑mediated breast cancer progression
To identify signaling pathways containing potential ther-
apeutic targets regulated by golgin-97, NGS and quan-
titative proteomic analysis were applied. Analysis of the
intersection of the two resulting datasets revealed that
783 DEGs were significantly upregulated, with a twofold
change in expression, in two golgin-97 KO cell lines (2–9
KO and 2–20 KO) compared with that in MDA-MB-231
cells (Fig. 2A). e IPA results suggested that several
upstream regulators and pathway networks, including
cytokines (IL-1β, TNF), growth factors (BMP4, AGT),
signal transduction factors (p38 MAPK), and transcrip-
tion factors (NF-κB-RelA), are significantly involved in
the upregulation of these 783 DEGs (Fig.2B and Table1).
e RT‒qPCR results verified that the expression lev-
els of three DEGs (IL-1β, IL-6, and MMP1) regulated
by the abovementioned key regulators were significantly
higher in golgin-97 KO cells than in MDA-MB-231 cells
(Fig. 2C). Additionally, DAVID bioinformatic analysis
revealed that the DEGs upregulated by golgin-97 KO
mediate biological processes such as the Wnt signaling
pathway, the MAPK cascade, wound healing, and the cel-
lular response to IL-1 (Fig.2D). We also performed TMT-
based quantitative proteomics to identify 7271 quantified
proteins in golgin-97 KO and MDA-MB-231 cells. Using
1.5 SDs of the fold change as a cutoff value, we identi-
fied 219 proteins upregulated (1.44-fold increase) by gol-
gin-97 KO. Consistent with the NGS analysis results, our
combined proteomic analysis/IPA revealed that TGFβ1,
EHF, TNF, JUNB, and IL-1α were the main activators
upregulated in response to golgin-97 KO (Fig. 2E and
Table S3). ese results were consistent with our ear-
lier study in which we utilized a quantitative proteomics
approach to establish a dataset that profiles the secreted
proteome, termed the secretome, which comprises 1,872
identified and quantified proteins from the conditioned
media of MDA-MB-231 cells, both with and without
golgin-97 knockdown. Among the proteins identified,
103 exhibited a 1.5-fold change in secretion levels. Of
these proteins, 88, including IL-6, IL-8, GM-CSF, TL1A,
PLAU, and ISG15, exhibited upregulation, whereas 15
proteins demonstrated downregulation in the golgin-
97-knockdown cells in comparison to the control cells.
Gene Ontology analysis revealed that these 103 proteins
were associated primarily with processes associated with
the wound response, cell migration, cellular localiza-
tion, cellular motility, and immune system functions (Fig.
S2). Our previous study also revealed that conditioned
medium from golgin-97-knockdown cells promoted
recipient cell migration and invasion, whereas an NF-κB
inhibitor reduced golgin-97-knockdown-induced TNBC
cell motility [17]. Taken together, these results suggest
that golgin-97 KO promotes cancer progression primar-
ily by modulating inflammatory responses and Wnt/
MAPK signaling in breast cancer cells.
Golgin‑97 KO induces tumor progression andmetastasis
accompanied bylung colonization andinammatory cell
inltration
In this study, we measured higher levels of murine TGFβ
and the proinflammatory cytokine TNFα in primary
tumors derived from golgin-97 KO cells than in those
derived from MDA-MB-231 cells (Fig. 3A), indicat-
ing that golgin-97 KO may promote tumor growth and
metastasis by modulating the tumor microenvironment.
Moreover, IHC analysis of primary tumors revealed
increased expression of the proliferation marker Ki67 and
the macrophage/monocyte marker CD68 in golgin-97
KO tumors (Fig.3B). To investigate whether golgin-97
influences macrophage polarization in the tumor micro-
environment of breast cancer, we conducted a co-culture
experiment involving cancer cells and PMA-induced
human leukemia monocytic THP-1 cells. Our results
indicated that both parental MDA-MB-231 cells and gol-
gin-97 KO cells induced a mix of pro-inflammatory and
anti-inflammatory factors production, enabling mac-
rophages to exhibit characteristics of both M1 and M2
polarization. is finding aligns with recent studies that
highlight the complexity of macrophage polarization,
where cells can display traits of both M1 and M2 pheno-
types [21, 22]. To further examine the effect of golgin-97
on macrophage polarization and function within the
tumor microenvironment associated with breast cancer
Page 6 of 18
Liuetal. Cell Communication and Signaling (2025) 23:22
Table 1 The IPA bioinformatics analysis reveals the upstream regulators for 783 up-regulated genes induced by golgin-97 knockout
Upstream regulator Molecule type p value Target molecules in dataset
IL1B Cytokine 1.00E-08 ACKR2,ADAMTS1,ANGPTL4,APOB,ASS1,BMF,C1R,C3,CABP1,CCL11,CCL20,CCL24,CEBPB,CHS
T6,CSF3,CXCL1,CYP19A1,CYP1B1,DUSP1,EBI3,ELF3,ENG,FPR2,GDF15,HAS1,HLAA,IER3,IER5L,
IL18,IL18R1,IL1A,IL1B,IL6,IL7R,INHBA,IRAK3,ITGB3,ITPKB,LCN2,LDHA,LOXL4,MAFA,MAPT,MEF
2B,MEFV,METRNL,MMP1,MMP3,MUC2,MYLK3,NOS3,PAPPA,PLXDC2,PTGS1,PTGS2,REN,SAA2
,SCX,SERPINB2,SERPINB3,SHH,SLC1A3,SOCS1,SRGN,ST18,TAP2,TGM2,TNFRSF11B,TREM1,VS
NL1,WNT5A,ZC3H12A
BMP4 Growth factor 1.15E-08 CCL11,CDH5,CEBPB,CSF3,CYP19A1,DIO2,DLX3,DLX4,ELF3,HSD3B1,IL6,INHBA,LEF1,LEFTY1,
MMP3,PGF,PITX2,PRKCH,PRR5,SHH,SNAI1,SPINT1,TAL1,TFAP2C,TGM2,TNFRSF11B,WNT5A
TNF Cytokine 1.16E-08 ACKR2,ADAMTS7,ALB,ALDH3A1,ALOX5AP,ANGPTL4,APOA1,AQP3,ASGR1,ASS1,B4GALNT
1,BDKRB2,BIK,BMP6,C1QTNF1,C3,CABP1,CCL11,CCL20,CCL24,CCN4,CD207,CD70,CDH11
,CDH5,CDO1,CEBPB,CIB2,CMKLR1,CSF3,CXCL1,CYP19A1,CYP1B1,DUSP1,DUSP14,DUSP9,
EBI3,ECH1,ELF3,ENG,FAT2,FBXO32,FPR1,FPR2,GDF15,GPRC5B,HAND1,HAS1,HCAR3,HEPA
CAM,HLAA,IER3,IL18,IL18R1,IL1A,IL1B,IL6,IL7R,INHBA,IRAK3,ITGB3,LAMP3,LCN2,LDHA,LPL,LR
IG1,LTBP2,MAFA,MEFV,METRNL,MMP1,MMP3,MUC2,MUC20,NFATC1,NOS3,PAPPA,PCK1,PDG
FA,PGF,PLA2G4C,PLXDC2,PTGS1,PTGS2,RAPGEF5,ROBO1,RRAD,SERPINB2,SHH,SLC16A2,SLC
1A3,SNAI1,SOCS1,SOX4,SYNGR3,TAPBP,TFAP2C,TG,TGM2,TH,TNFRSF11B,TNNC1,TREM1,TRI
M63,UCN2,VIPR1,WNT1,WNT3A,WNT5A,ZC3H12A,ZNF750
P38 MAPK Group 2.72E-08 ASS1,CCL11,CD70,CDH5,CEBPB,CSF3,CXCL1,CYP19A1,DIO2,DUOX2,DUSP1,FBXO32,FGF21,I
ER3,IL1A,IL1B,IL6,INHBA,ITGB3,MAL,MMP1,MMP3,MUC2,MYBPH,NFATC1,NOS3,PCK1,PTGS2,
RRAD,SERPINB2,SNAI1,TH,TJP2,TNNC1,TREM1,WNT5A
DSCAML1 Other 5.51E-08 ATOH7,BMP6,DNER,EMX1,LHX9,MAPT,NPAS3,NRCAM,NTNG2,PAX5,PCDH17,PDGFA,PRKCH,
ROBO2,SHANK1,TUBB4A
NFkB1-RelA Complex 6.06E-08 CCL11,CCL20,CSF3,CXCL1,IL1B,IL6,LCN2,MMP1,MMP3,PTGS2,TNFRSF11B
AGT Growth factor 6.53E-08 ADGRE1,AMBP,ANGPTL8,BDKRB2,BMP6,C1QTNF1,C3,CCN4,CDH5,COL4A1,COL4A2,COL6
A2,COLEC10,CYP19A1,DIO2,DUSP1,ENG,FGA,GAA,GABBR1,GAS2,GDF15,HSD3B1,IL18,IL
18R1,IL1A,IL1B,IL6,ITGB3,ITPKB,LCN2,LGI3,LOXL4,LRP1,LRRC7,LTBP2,LTBP3,MAPT,MMP1,MMP
3,MYO7A,NOS3,PDGFA,PGF,PTGS1,PTGS2,PTPN22,REN,ROBO1,SCG2,SERPINB2,SH2D1B,SLC1
A1,SOCS1,SOST,SOX4,SPINT1,SUSD5,TH,TNFRSF11B,VTN,XIRP1
Dexa-methasone Chemical drug 9.83E-08 ABCC6,ACY3,ADAMTS1,AFP,ALB,ALOX5AP,ANGPTL4,APOA1,ATF7IP2,ATP9A,AVPR1B,BDKRB2
,BMP5,C12orf42,C3,C8A,CCL11,CCL20,CDH11,CDKN1C,CEBPB,CELF2,CLDN9,CMKLR1,COL4
A1,COL4A2,COL6A2,COL7A1,CTSE,CTSW,CXCL1,CYP19A1,CYP1B1,DIO2,DNER,DUSP1,EDA,F
AM107A,FBLN1,FBXO32,FGA,FGF21,FOXQ1,FPR2,FSD2,GABBR1,GAS2L2,GBP5,GDF15,GNGT
2,GNRH2,HCST,HLAA,HSPA6,IER3,IL18,IL18R1,IL1A,IL1B,IL2RB,IL6,IL7R,INHBA,IRAK3,ITGB3,KC
NK3,KLRC4KLRK1/KLRK1,LCN2,LDHA,LEF1,LEFTY1,LPL,LRP1,LSR,LTBP2,MATN1,MATN3,MMP
1,MMP3,MUC2,NFATC1,NOS3,NPPC,NTF3,OR51E1,OSBP2,PCK1,PDGFA,PPP1R14C,PTGFRN,P
TGS1,PTGS2,PTPN22,RAB37,RPS6KA2,RRAD,SAA2,SAA4,SCG2,SERPINB2,SHH,SLC1A3,SLC2A
5,SLC4A1,SNAI1,SOCS1,SRGN,TAP2,TGM2,TLE2,TMEM61,TNFRSF11B,TNS4,TRIM63,TRPM2,T
UBB1,UCN2,VIPR1,WNT2,WNT5A,ZC3H12A,ZIC2,ZNF750
DSCAM Other 1.21E-07 EMX1,KCNJ4,MAPT,NPAS3,NRCAM,NTF3,NTNG2,PAX5,PCDH17,PDGFA,PRKCH,ROBO2,SLC1A
1,SYNGR3,TAL1,TUBB4A
CD36 Transmembrane receptor 2.19E-07 ADGRE1,APOA1,APOB,CCL20,COL4A1,CSF3,CXCL1,IL1A,IL1B,IL6,LPL,LRP1,MMP1,MMP3,SER
PINB2,TNFRSF11B
Fig. 2 Multi-omics reveals the involvement of key factors in golgin-97 KO-mediated breast cancer progression. A The DEGs between the two
golgin-97 KO cell lines and the MDA-MB-231 cells were analyzed via next-generation sequencing (NGS). The Venn diagram shows the 783
overlapping genes upregulated by golgin-97 KO. B The graphical summary of the IPA results shows the key molecules and functions of these 783
upregulated genes in golgin-97 KO cells. C qRT‒PCR analysis of IL-1β, IL-6, and MMP1 mRNA levels in MDA-MB-231 and golgin-97 KO cells. The data
are shown as the means ± SDs. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 based on one-way ANOVA. D The biological processes of the 783 genes
upregulated by golgin-97 KO were analyzed with the DAVID bioinformatics resource. E Quantitative proteomics combined with bioinformatic
analysis revealed network and disease-related functions in which the 219 proteins upregulated by golgin-97 KO were involved. The orange
octagons, squares, and ovals represent functions, cytokines, and transcriptional regulators, respectively. The orange hourglasses represent canonical
pathways. The orange dotted lines indicate activation, and the gray dotted lines indicate unpredicted effects
(See figure on next page.)
Page 7 of 18
Liuetal. Cell Communication and Signaling (2025) 23:22
Fig. 2 (See legend on previous page.)
Page 8 of 18
Liuetal. Cell Communication and Signaling (2025) 23:22
invivo, we performed an association analysis of golgin-97
gene expression (GOLGA1) with M1 and M2 mac-
rophages in BRCA-Basal (TNBC > 70%) via TIMER2.0
(http:// timer. cistr ome. org/), an integrated online data-
base that compiles cancer research and immune associa-
tion data from multiple sources. Our analysis suggested
that golgin-97 may be positively correlated with M1
macrophages, as indicated by the CIBERSORT data, but
negatively correlated with M2 macrophages, accord-
ing to findings from the TIDE data (Fig. S3). Together,
these observations suggest that the downregulation
of golgin-97 in breast cancer cells may increase M2
Fig. 3 Golgin-97 KO induces tumor progression and metastasis accompanied by lung colonization and inflammatory cell infiltration. A qRT‒PCR
analysis of murine IL-6, TNFα, and TGFβ mRNA levels in primary tumors from mice (n = 9 mice per group). The data are based on an unpaired t test. B
Representative images of IHC staining for Ki67 and CD68 in primary tumors. Scale bars, 100 μm. C Representative images of lung metastasis sections
with H&E staining and IHC staining with anti-Ki67 and anti-CD68 antibodies. The yellow arrows delineate the tumor region. Scale bars, 200 μm (H&E
and Ki67) and 50 μm (Ki67 and CD68). D‑F Quantification of lung metastatic nodules, Ki67-positive cells, and CD68-positive cells in the MDA-MB-231
and 2–9 KO groups (n = 4 mice per group). The quantitative data were analyzed via an unpaired t test. The data are presented as the means ± SDs
(*p < 0.05; **p < 0.01; ****, P < 0.0001)
Page 9 of 18
Liuetal. Cell Communication and Signaling (2025) 23:22
macrophage polarization through complex communi-
cation between multiple cell types in tumor microen-
vironment. Next, we examined whether golgin-97 KO
promotes metastatic tumor cell colonization. H&E stain-
ing and Ki67 and CD68 expression were evaluated in lung
sections. Quantitative analysis revealed that golgin-97
KO significantly increased the nodule number and lung
metastatic colonization, as well as the infiltration of mac-
rophages/monocytes (Fig.3C-F). ese results suggested
that golgin-97 KO significantly and positively influences
primary tumor colonization and metastasis, an effect
that involves proinflammatory factor-controlled interplay
between golgin-97 KO cells and stromal cells.
Targeting ERK/MAPK kinase attenuates golgin‑97
KO‑induced breast cancer malignancy andenhances
paclitaxel treatment ecacy
To search for new therapeutic targets to benefit patients
with malignancy and chronic inflammation induced by
low expression of golgin-97, we conducted an IPA of the
783 upregulated DEGs identified from the intersection
of NGS data derived from two golgin-97 KO cell lines.
e analysis revealed a total of 629 chemical compounds,
including both activators and inhibitors. Notably, when
the predicted activation state of inhibition was examined,
36 chemical compounds were identified and subsequently
ranked on the basis of a p value of less than 0.05, with
the top five compounds documented in Table2. Among
these, we emphasized two leading chemical inhibitors:
U0126 (MEK1/2 inhibitor) and SB203580 (p38 MAPK
inhibitor). A wound healing assay revealed that gol-
gin-97 KO cells had greater collective migration capac-
ity than did MDA-MB-231 cells. Importantly, SB203580
and U0126 alone or in combination significantly reduced
golgin-97 KO-induced cell motility (Fig.4A). RT‒qPCR
analysis revealed that U0126 and SB203580 significantly
reduced the mRNA expression levels of IL-1β, IL-6, and
MMP1 in golgin-97 KO cells (Fig. 4B). Gene-specific
silencing via siRNA transfection was further used to con-
firm the phenomena shown by chemical inhibitor treat-
ment. As shown in Fig.4C, the mRNA expression levels
of IL-1β, IL-6, and MMP1 were decreased by p38 MAPK
knockdown. ERK1/2 knockdown reduced the mRNA
expression of IL-1β and MMP1 but not that of IL-6.
Knockdown of p38 MAPK and ERK1/2 also significantly
inhibited the increased cell migration ability induced by
golgin-97 depletion in MDA-MB-231 and MCF-7 cells
(Fig.4D-E), and the knockdown efficacy is shown in Fig.
S4A-B. Although golgin-97 KO did not promote the pro-
liferation of breast cancer cells invitro, treatment with
U0126 alone or in combination with SB203580 reduced
the viability of golgin-97 KO cells and MDA-MB 231
cells after 48 h and 72 h, respectively (Fig.4F). Paclitaxel
(PTX) is the most commonly used chemical agent for
the clinical treatment of TNBC, and long-term chemo-
therapy causes severe side effects in patients [23, 24]. We
then tested whether treatment with U0126 or SB203580
as adjuvants with paclitaxel would benefit patients with
golgin-97 deficiency. e results of the cell viability
assay revealed that treatment with PTX or two-kinase
inhibitors (U0126 and SB203580) was cytotoxic to gol-
gin-97 KO cells (Fig.4G). Notably, combined treatment
with these three drugs (U0126 + SB203580 + PTX) sig-
nificantly resulted in a synergistic effect compared with
PTX alone or the combination of U0126 and SB203580
(Fig.4G). ese results suggest that the inhibition of both
the ERK1/2 kinase pathway and the p38 MAPK pathway
has therapeutic potential for the treatment of breast can-
cer patients with low expression of golgin-97.
Inhibition ofERK/MAPK kinase iseective inovercoming
golgin‑97 KO‑induced tumor growth andlung metastasis
We next examined the therapeutic potential of ERK1/2
kinase and p38 MAPK inhibitors in an orthotopic mouse
model. We observed that treatment with PTX alone, two-
kinase inhibitors, or a combination of the three drugs
significantly reduced the tumor growth rate and tumor
volume, with targeting of ERK1/2 kinase and p38 MAPK
enhancing the efficacy of PTX-based chemotherapy.
(Fig.5A-B). Specifically, growth inhibition and survival
protection were most pronounced in the kinase inhibi-
tor treatment groups (Fig. 5B-C). To confirm whether
Fig. 4 Targeting ERK/MAPK kinase attenuates golgin-97 KO-induced breast cancer malignancy and enhances paclitaxel treatment efficacy. A
Wound healing assay of MDA-MB-231 and 2–9 KO cells treated with vehicle (veh.) control, SB203580 (20 μM), U0126 (20 μM), or SB203580 (20
μM) + U0126 (20 μM) for 17 h (left panel) and quantification of cell migration (right panel). The quantitative data were analyzed via one-way ANOVA.
qRT‒PCR analysis of IL-1β, IL-6, and MMP1 mRNA levels in 2–9 KO cells treated with inhibitors (B) or gene-specific knockdown (C) as indicated.
The data were analyzed via one-way ANOVA. Wound healing assays with gene-specific knockdown of p38 MAPK and ERK1/2 as indicated were
performed on 2–9 KO cells (D) and MCF-7 cells (E). The data were analyzed via one-way ANOVA. F MDA-MB-231 and 2–9 KO cells were treated
with vehicle control, SB203580, U0126, or a combination (SB203580 + U0126) for the indicated times, followed by a cell viability assay. G 2–9 KO
cells were treated with vehicle control, SB203580 + U0126, PTX, or PTX + SB203580 + U0126 for the indicated times, followed by a cell viability assay.
The data were analyzed via two-way ANOVA. The data are presented as the means ± SDs. *p < 0.05; **p < 0.01; ***, P < 0.001; ****, P < 0.0001. Images
from each group in all the wound healing assays were acquired in three random fields of view via microscopy
(See figure on next page.)
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Liuetal. Cell Communication and Signaling (2025) 23:22
Fig. 4 (See legend on previous page.)
Page 11 of 18
Liuetal. Cell Communication and Signaling (2025) 23:22
these two kinase inhibitors modulate cancer-associated
inflammation in this mouse model, the expression of
CD68 and inflammatory mediators in primary tumors
was examined. IHC revealed that PTX (PTX alone
and three drugs), but not two-kinase inhibitor, signifi-
cantly reduced CD68 expression (Fig. 5D-E). However,
qRT‒PCR revealed that the administration of kinase
inhibitors alone or in combination with PTX resulted in
reduced mRNA expression of murine TNFα and TGFβ
(Fig. 5F-G). Compared with that in the vehicle group,
the expression of murine TGFβ but not TNFα was lower
in the PTX alone group (Fig.5F-G). Additionally, quan-
titative analysis of IHCs obtained from lung tissue sec-
tions revealed that treatment with two-kinase inhibitors
(two-kinase inhibitors alone or three drugs) but not PTX
alone resulted in lower levels of human Ki67 expression,
indicating a reduction in lung metastatic colonization
(Fig.5H-I). Notably, we observed significant infiltration
of CD68 and induction of cleaved caspase-3 expres-
sion in lung tissues treated with PTX alone or the three
drugs, suggesting that PTX treatment may increase the
potential for lung inflammation and lung injury (Fig.5J-
L). ese results collectively suggest that ERK1/2 and
p38 MAPK inhibitors can effectively treat golgin-97-KO-
induced breast cancer malignancy by reducing inflamma-
tion at the primary tumor site and inhibiting lung organ
metastasis.
Hypoxia mimetics andERK/MAPK signaling regulate
golgin‑97 expression inbreast cancer
Hypoxia is detected in most solid tumors and strongly
promotes tumor growth and survival through metasta-
sis [25–29]. Interestingly, the activation of the hypoxia-
inducible factor HIF1α/heme oxygenase-1 signaling
pathway attenuated sepsis-induced acute lung injury and
reduced Golgi stress by increasing the expression of Golgi
structural proteins such as golgin-97 [30]. We then exam-
ined whether HIF1α influences golgin-97 and MAPK
signal transduction in breast cancer cells. We detected
HIF1α accumulation with increasing doses of CoCl2 but
decreased protein levels of golgin-97, TGN46, and IκBα
(Fig. 6A-D). Upon incubation with CoCl2, ERK1/2 and
Table 2 Potential chemical inhibitors used to suppress the 783 genes upregulated by golgin-97 knockout
Upstream regulator Molecule type Predicted
activation
state
p value Target molecules in dataset
U0126 Chemical - kinase inhibitor Inhibited 6.34E-06 ADGRE1,ANGPTL4,AQP3,BMP6,C3,CCL11,CCN4,CDH5,CDKN1C,CEBPB,COL
4A1,CSF3,CXCL1,CYP19A1,CYP1B1,DUSP1,GDF15,IER3,IL1A,IL1B,IL6,INHBA
,ITGA2B,LPL,MMP1,MMP3,MUC2,MYO7A,NFATC1,NXPH4,PTGS2,REN,SERPI
NB2,SHH,SOCS1,TAP2,TGM2,TH,TNFRSF11B,ZIC2
SB203580 Chemical - kinase inhibitor Inhibited 2.38E-05 ALOX5AP,C3,CCL11,CCL20,CD207,CEBPB,CXCL1,CYP1B1,DUOX2,DUSP1,E
BI3,FGF21,FOXQ1,FPR2,HAND1,HAS1,HCAR3,HEPACAM,IER3,IL18,IL1A,IL1
B,IL6,ITGB3,MMP1,MMP3,MUC2,NOS3,PCK1,PDGFA,PTGS2,SNAI1,TGM2,TN
FRSF11B,TREM1
PD98059 Chemical - kinase inhibitor Inhibited 4.43E-05 ALOX5AP,APOA1,BDH1,C3,CCL11,CCL20,CEBPB,COL4A1,CXCL1,CYP19A1,C
YP1B1,DIO2,DUSP1,ENG,FPR2,HAS1,IER3,IL1A,IL1B,IL6,ITGA2B,ITGB3,KLRC2
,LDHA,LPL,MMP1,MMP3,MT1F,MUC2,NOS3,PCK1,PDGFA,PTGS2,REN,SERPI
NB2,SHH,SNAI1,TH,TP73
Aspirin Chemical drug Inhibited 1.17E-04 APOA1,BMP6,CDH5,CSF3,CXCL1,ENG,GDF15,IER3,IL18,IL1A,IL1B,IL6,ITPKB,
MMP1,PTGS1,PTGS2,SOX4
AS1842856 Chemical reagent Inhibited 1.20E-04 CXCL1,FBXO32,IL6,MMP1,MMP3,TRIM63
(See figure on next page.)
Fig. 5 Inhibition of ERK/MAPK kinase is effective in overcoming golgin-97 KO-induced tumor growth and lung metastasis. A NOD/SCID mice were
subcutaneously injected with 2-9 KO cells, followed by veh. control, inhibitor (SB203580+U0126), PTX, or a combination of an inhibitor with PTX
(PTX+SB203580+U0126), and tumor growth was monitored. The tumor sizes (cm) are indicated at the bottom. B Chart showing tumor sizes
and weights from 5A. The data are based on two-way ANOVA. C Survival rates of NOD/SCID mice from 5A. Kaplan–Meier plots in which the standard
tumor size (300 mm3) was used as a criterion for euthanasia in the veh.-control and drug treatment groups. D Representative images of IHC staining
for CD68 in primary tumors. Scale bars, 50 μm. E Quantification of CD68-positive cells derived from panel 5D. F‒G qRT‒PCR analysis of murine
TNFα and TGFβ mRNA levels in primary tumors from mice. The data were analyzed via one-way ANOVA. H and J Representative images of lung
metastasis sections with H&E staining and IHC staining with anti-Ki67 and anti-CD68 antibodies. The yellow arrows delineate the lung metastasis
region of the primary tumor. Quantification of Ki67-positive cells (I) and CD68-positive cells (K) from the lower panel (5H and 5J). The data were
analyzed via one-way ANOVA. L Western blotting analysis of cleaved caspase-3 and pro-caspase-3 protein levels in the lung tissues of the mice.
Actin was used as the internal control. The quantification of protein expression is shown in the lower panel. The data are presented as the means ±
SDs (*p < 0.05; **p < 0.01; ***, P < 0.001; ****, P < 0.0001)
Page 12 of 18
Liuetal. Cell Communication and Signaling (2025) 23:22
Fig. 5 (See legend on previous page.)
Page 13 of 18
Liuetal. Cell Communication and Signaling (2025) 23:22
p38 MAPK were activated following the decrease in gol-
gin-97 protein expression (Fig. 6A, E-F). ese results
suggest that, unlike acute lung injury, hypoxia may be a
physiological condition that contributes to the down-
regulation of golgin-97 expression and the activation
of ERK/MAPK signaling in cancer cells. Interestingly,
treatment of MDA-MB-231 cells with U0126 alone or
with two inhibitors (SB203580 + U0126) for 24 h caused
a significant increase in golgin-97 protein expression in
the MDA-MB-231 cells (Fig. 6G-H). A slight increase
(approximately 20%) in golgin-97 mRNA levels was
observed in combination-treated cells, suggesting an
unexpected mechanism by which MAPK signaling regu-
lates golgin-97 expression at the protein level (Fig. S5A).
Consistent with these findings, we also observed an
increase in golgin-97 protein but not golgin-97 mRNA
expression with single knockdown of ERK1/2 and double
knockdown of ERK1/2 and p38 MAPK in MDA-MB-231
and MCF-7 cells (Fig. 6I-L and S5B-3C). ese results
support the hypothesis that a potential negative regula-
tory loop exists between ERK/MAPK signaling and gol-
gin-97 expression under hypoxic conditions; therefore,
the inhibition of ERK/MAPK may abrogate the tumor-
promoting pathways induced by golgin-97 deficiency
(Fig.6M).
Discussion
Accumulating evidence indicates that the Golgi appara-
tus is a critical regulator of tumor progression. Coiled-coil
golgins play a central role in controlling membrane traf-
ficking and act as regulators of Golgi integrity and signal
transduction [8, 10, 11, 31, 32]. For example, dysregula-
tion of Golgi-localized Cdc42 signaling by the cis-Golgi
matrix protein GM130 may promote the progression of
colon and breast cancer [33, 34]. Our recent studies with
Kaplan‒Meier plotter (http:// kmplot. com) and Oncomine
(https:// www. oncom ine. org) data revealed that low gol-
gin-97 expression is correlated with poor survival and
increased invasiveness in breast cancer cells [17]. On
the basis of the cSurvival database (https:// tau. cmmt.
ubc. ca/ cSurv ival/)) and the UALCAN database (https://
ualcan. path. uab. edu/), we also observed that low gol-
gin-97 expression significantly worsened the progno-
sis of progression-free survival (P = 0.0058). TCGA data
revealed that golgin-97 expression is notably lower in
TNBC patients than in normal controls (P < 1 × 10–12),
luminal cancer patients (P = 1.11 × 10–16), and HER2-
positive patients (P = 7.39 × 10–4) (Fig. S6). ese results
confirm that low golgin-97 expression is a key feature of
TNBC and is linked to poor clinical outcomes. e cur-
rent study showed that golgin-97 KO can promote the
metastatic dissemination and lung colonization of TNBC
cells in vivo (in zebrafish and immunodeficient mice).
Our orthotopic mouse model studies further confirmed
that golgin-97 KO has a positive regulatory effect on pri-
mary tumor growth and distant metastasis, accompanied
by a proinflammatory tumor microenvironment with
features such as increased secretion of murine tumor-
associated macrophage-related factors in tumor tissues
(Fig. 6M). Macrophages and growth factors/cytokines
directly contribute to processes facilitating breast tumor
progression and metastasis, including tumor cell growth,
migration, invasion, and intravasation [35, 36]. To assess
whether inflammatory cytokines or growth factors mod-
ulate golgin-97 expression, we administered IL-6, IL-1β,
and TGF-β to MDA-MB-231 cells. e results demon-
strated that short-term exposure to these cytokines led
to a decrease in golgin-97 mRNA expression. Prolonged
stimulation with TGF-β alone sustained the reduc-
tion in golgin-97 mRNA levels, whereas treatment with
IL-6 and IL-1β tended to lead to recovery of golgin-97
mRNA expression. Nonetheless, golgin-97 protein levels
increased following stimulation with all three cytokines
(Fig. S7). ese findings suggest a complex regulatory
mechanism involving golgin-97 in response to inflamma-
tory cytokines. Specifically, while short-term exposure to
IL-6, IL-1β, and TGF-β reduces golgin-97 mRNA levels,
the simultaneous increase in golgin-97 protein expres-
sion indicates a potential posttranscriptional regulatory
effect. is discrepancy may reflect an adaptive cellular
response in which cells prioritize the production or sta-
bilization of golgin-97 protein despite reduced mRNA
Fig. 6 Hypoxia mimetics and ERK/MAPK signaling regulate golgin-97 expression in breast cancer. A MDA-MB-231 cells were treated with increasing
concentrations of CoCl2 for 24 h and then subjected to Western blotting analysis with phosphosite- and protein-specific antibodies, as indicated.
Actin was used as the internal control. B-F Quantification of protein expression, as shown in panel 6A. G MDA-MB-231 cells treated with kinase
inhibitors were prepared for Western blotting analysis via phosphosite- and protein-specific antibodies, as indicated. Actin was used as the internal
control. H Quantification of golgin-97 protein expression levels from panel 6G. The quantitative data were analyzed via one-way ANOVA. The
golgin-97 protein level in MDA-MB-231 cells (I) and MCF-7 cells (J) following p38 MAPK, ERK1/2 individual knockdown, or combined with double
knockdown. K, L Quantification of golgin-97 protein expression levels from panel 6I‒6J. The data were analyzed via one-way ANOVA. The data are
presented as the means ± SDs. *p < 0.05; **p < 0.01; ***, P < 0.001 based on one-way ANOVA. M Golgin-97 deletion promotes cancer progression,
an effect that involves hypoxia, MAPK signaling, and the tumor microenvironment. MAPK inhibitors could be used to suppress golgin-97
deregulation in TNBC. This model is based in part on an illustration from biorender.com
(See figure on next page.)
Page 14 of 18
Liuetal. Cell Communication and Signaling (2025) 23:22
Fig. 6 (See legend on previous page.)
Page 15 of 18
Liuetal. Cell Communication and Signaling (2025) 23:22
levels, possibly to support functions associated with
cellular stress and inflammation. Given that neither
the knockdown nor the KO of golgin-97 significantly
increased cell proliferation in vitro, we propose that
tumor cell–stromal cell interactions have a critical impact
during golgin-97 KO-mediated cancer progression.
Currently, drugs targeting Golgi-resident proteins for
treating various cancers and metastases have entered
clinical trial stages, demonstrating the feasibility and
potential of targeting the Golgi apparatus in cancer ther-
apy [37]. However, the challenges remain the specificity of
the target, with a significant reduction in complications.
Our omics study revealed that the Wnt signaling path-
way, MAPK kinase cascades, and inflammatory cytokines
are the key regulators that are significantly upregulated
in golgin-97 KO cells. Previous studies have reported
that individual treatment with the ERK inhibitor U0126
or the p38 MAPK inhibitor SB203580 effectively inhibits
the proliferation and invasion of breast cancer cells, but
the specific regulatory factors involved remain unclear
[38–43]. During cancer progression, activated mac-
rophages can polarize into two major subsets, M1 and
M2, which promote tumorigenic capabilities through the
secretion of inflammatory cytokines, including TNFα
and TGFβ [44]. Although combined treatment with
U0126 and SB203580 did not decrease CD68 infiltra-
tion in mice derived from golgin-97 KO cells, these two
kinase inhibitors significantly reduced murine TNFα and
TGFβ expression levels in primary tumors. e current
study proposes an intriguing and promising therapeutic
strategy: administering ERK/MAPK inhibitors to main-
tain tumor-suppressing golgin-97 protein expression in
TNBC, thereby attenuating breast cancer growth, the
inflammatory response, and lung metastasis. Our study
also revealed the benefits of ERK/MAPK inhibitors in
preventing lung metastasis and lung injury. Specifically,
pulmonary drug toxicity is a cause of interstitial lung
disease, which can range from benign infiltrates to life-
threatening acute respiratory distress syndrome. Direct
dose-dependent toxicity and immune inflammation are
the main causes of lung damage and fibrosis. We showed
that PTX alone but not kinase inhibitors alone caused
CD68 infiltration into lung sections and induced lung
damage. Severe lung damage occurred when PTX was
given in combination with these two inhibitors, suggest-
ing that the drug dosage and/or other unknown signal-
ing pathways contribute to this lung damage. Future work
will focus on using mouse breast cancer animal models
to reveal the interaction between golgin-97 and specific
immune cells and optimize combination treatment strat-
egies for breast cancer.
U0126 is a MEK1/2 inhibitor that primarily targets
MEK in the MAP kinase pathway, blocking downstream
ERK1/2 signaling and impacting p38 MAPK activity [45].
Notably, low-dose, short-term use of U0126 reduces p38
MAPK phosphorylation, whereas prolonged treatment
with both SB203580 and U0126 results in increased phos-
phorylation of p38 and ERK1/2. is suggests a recipro-
cal balance between the phosphorylation of ERK and p38,
where the inhibition of one pathway leads to an increase
in the activity of the other pathway [46, 47]. For exam-
ple, treatment with high concentrations of SB203580 has
been reported to activate Raf-1 and ERK signaling [48,
49], supporting potential crosstalk between ERK and p38
MAPK signaling with SB203580 in cancer therapy. U0126
may inhibit breast cancer growth by blocking the cell
cycle, but the role of p38 in cancer remains complex and
is currently a subject of ongoing debate [45, 50]. Impor-
tantly, our findings support this complexity, aligning
with the results presented in Fig.6G and Fig.4C, which
demonstrated potential crosstalk between U0126 and
SB203580. is interaction also strengthens the idea that
the combination of these two kinase inhibitors results
in antitumor activity invivo (Fig. 5) while also helping
to explain the lack of synergistic effects observed with
U0126 and SB203580 in our invitro assays (Fig.4).
Our results demonstrated that U0126 and SB203580
individually inhibited the mRNA expression of inflam-
matory cytokines and MMP1, which was upregulated by
golgin-97 KO. e increased IL-6 mRNA levels in 2–9
KO cells after ERK knockdown, as shown in Fig. 4C,
may be due to several factors. First, there is a reciprocal
relationship between the phosphorylation of ERK and
p38 MAPK, where inhibiting one pathway may increase
the other [46, 47]. Second, p38 MAPK and IL-6 have a
reciprocal and interconnected relationship in several
pathways [51]. ird, Wnt5a activates JNK, ERK1/2, and
ERK5, contributing to IL-6 expression in preadipocytes
[52]. However, loss of ERK1/2 can stimulate ERK5 sign-
aling in epithelial cells, enhancing IL-6 expression in
the tumor microenvironment [53]. We noted increased
Wnt5a in golgin-97 KO cells (data not shown), poten-
tially increasing ERK5 activity and IL-6 mRNA levels.
erefore, the increase in IL-6 following ERK knock-
down is plausible. Compared with U0126, SB203580
alone was insufficient to reduce cell viability but more
effectively inhibited the collective migration of golgin-97
KO cells, suggesting that different MAPK signaling path-
ways may be required for golgin-97 KO-induced cell
proliferation and motility (Fig.6M). e siRNA knock-
down results suggest that p38 MAPK and ERK1/2 not
only impact golgin-97 protein expression but also modu-
late cell motility and the cytotoxic effects of chemother-
apy. However, we found that SB203580 treatment alone
did not increase golgin-97 protein expression, which is
Page 16 of 18
Liuetal. Cell Communication and Signaling (2025) 23:22
inconsistent with p38 silencing. is inconsistency may
be due to the specificity and off-target effects of kinase
inhibitors [54]. Treatment with high concentrations
of SB203580 has been reported to activate Raf-1 and
ERK signaling [48, 49], supporting potential crosstalk
between ERK and p38 MAPK signaling with SB203580
in cancer therapy. Consistently, we also observed that
combined treatment with U0126 and SB203580 had the
most promising effects on cancer progression in gol-
gin-97 KO cells.
We observed that the suppression of p38 MAPK
and ERK1/2 increased the protein but not the mRNA
expression of golgin-97. ERK reportedly promotes
tumorigenesis by regulating protein stability through
the ubiquitin‒proteasome pathway [55]; thus, MAPK
inhibitors may maintain the protein expression of gol-
gin-97 in breast cancer cells. Consistent with this idea,
the golgin-97, IκBα, and TGN46 protein levels were
reduced, but ERK and p38 MAPK were activated in
CoCl2-treated cells, indicating an inverse correlation
between golgin-97 expression and MAPK signaling
activity (Fig. 6M). Given that CoCl2-induced HIF1α
expression occurs mainly through ERK and p38 MAPK
activation [56, 57], we cannot exclude the possibility
that ERK and p38 MAPK signaling may be upstream
regulators of golgin-97. ese findings also support
the possibility of a feedback loop between MAPKs and
golgin-97 in the modulation of breast cancer progres-
sion under hypoxic conditions. Additionally, we found
that IL-1β levels were increased in golgin-97 KO cells.
IL-1β mainly regulates cell migration by activating p38
MAPK, and its substrate MAPKAPK2 is involved in
modulating the tumor microenvironment, thereby fur-
ther promoting the invasion of breast cancer cells [58,
59]. IL-1β-induced migration of MDA-MB-231 cells
under hypoxic conditions has also been reported [60].
By using a hypoxic chamber, we found that the protein
levels of golgin-97 were increased in MDA-MB231 cells
under short-term hypoxia treatment (15 min), whereas
golgin-97 and TGN46 were significantly decreased after
2 to 8 h of hypoxic exposure (data not shown). e cur-
rent study reports the interplay between hypoxia and
the golgin-97 and MAPK signaling pathways. However,
further studies are needed to determine whether this
mechanism is directly mediated by golgin-97 in vivo.
Nevertheless, we hypothesize that hypoxia and expo-
sure to unidentified stimulators are the physiological
conditions involved in golgin-97 downregulation, which
modulates the tumor microenvironment and promotes
cell aggressiveness invivo.
To our knowledge, our study is the first to report that
golgin-97 affects tumor growth and metastasis possibly
through communication between cancer cells and inflam-
matory stromal cells. Both the anti-inflammatory effects
and the restoration of tumor-suppressive golgin-97 expres-
sion contribute to the efficacy of U0126 and SB203580
kinase inhibitors in breast cancer therapy. Compared with
the paclitaxel regimen, these two kinase inhibitors have
the potential to prevent lung injury. Although golgin-97
does not have kinase activity or directly affects protein
stability, we propose that it may indirectly affect signaling
pathways such as the ERK1/2 and p38 MAPKpathways for
three reasons. First, golgin-97 aids in transport and organi-
zation within the TGN, helping to sort signaling molecules
and receptors [61]. By ensuring correct localization, gol-
gin-97 can alter protein activation in signaling pathways.
Second, as a tethering factor, it promotes vesicle fusion
and affects access to growth factor or cytokine receptors,
such as IL-10 [62, 63]. ird, changes in Golgi integrity,
especially in cancer cells, can disrupt the processing and
glycosylation of signaling molecules, significantly alter-
ing receptor signaling and activation of the ERK1/2 and
p38 MAPKpathways [64]. Investigations of the impact of
golgin-97 on different cancers are rare, and more evidence
is needed to explore its clinical application. Overall, this
study provides new therapeutic strategies for TNBC and
may benefit drug development.
Abbreviations
TNBC Triple-negative breast cancer
KO Knockout
PTX Paclitaxel
CoCl2 Cobalt chloride
DMSO Dimethyl sulfoxide
CCK-8 Cell Counting Kit-8
MAPK Mitogen-activated protein kinase
TNF Tumor necrosis factor
TGN Trans-Golgi network
ERK Extracellular signal-regulated kinase
MEK MAPK/extracellular signal-regulated kinase
HIF1α Hypoxia-inducible factor 1 alpha
IL-1β Interleukin 1 beta
IL-6 Interleukin 6
TGFβ Transforming growth factor beta
MMP-1 Matrix metalloproteinase-1
NF-κB Nuclear factor kappa-B
qPCR Quantitative PCR; RT‒PCR, reverse transcription polymerase
chain reaction
TMT Tandem mass tag
2D LC‒MS/MS Two-dimensional liquid chromatography‒tandem mass
spectrometry
NGS Next-generation sequencing
IPA Ingenuity Pathway Analysis
DEGs Differentially expressed genes
DAVID Database for Annotation, Visualization and Integrated
Discovery
WT Wild-type
SDs Standard deviations
Page 17 of 18
Liuetal. Cell Communication and Signaling (2025) 23:22
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s12964- 024- 02010-0.
The online version contains Appendix S1, Supplementary Figures S1-S7, and
Supplementary Tables S1-S3. All the data are contained within the article and/
or the supporting information. The mass spectrometry proteomics data have
been deposited to the ProteomeXchange Consortium via the PRIDE partner
repository [65] with the dataset identifier PXD056499.
Additional file 1.
Additional file 2.
Additional file 3.
Acknowledgements
We thank Ke-Wei Lin for the technical support in our genomic analysis. We
also appreciate Professor Chia-Rui Shen for providing the essential reagents for
the co-culture of cancer cells with monocytic cells.
Authors’ contributions
Y-C Liu contributed to the design of the experiments, performed the research,
analyzed the results, wrote the manuscript draft and revised the manuscript;
T-J Lin performed the research, analyzed the results and wrote the manuscript
draft; K-Y Chong contributed to the design of the experiments and performed
the research; G-Y Chen performed the research and analyzed the results; C-Y
Kuo performed the research and analyzed the results; Y-Y Lin performed the
research and analyzed the results; C-W Chang helped establish the zebrafish
model; T-F Hsiao performed the proteomic analysis; C-L Wang provided the
methodology; Y-C Shih performed the research and contributed to the data
analysis; and C-J Yu was responsible for conceptualization, experimental
design, supervision, writing, review & editing, and funding acquisition.
Data availability
The mass spectrometry proteomics data have been deposited to the Pro-
teomeXchange Consortium via the PRIDE partner repository with the dataset
identifier PXD056499 (http:// www. ebi. ac. uk/ pride).
Declarations
Ethics approval and consent to participate
The animal protocol was reviewed and approved by the Laboratory Animal
Use Committee of Chang Gung University (CGU107-228).
Competing interests
The authors declare no competing interests.
Author details
1 Department of Cell and Molecular Biology, College of Medicine, Chang Gung
University, 259 Wen-Hwa 1 road, Guishan District, Taoyuan, Taiwan. 2 CardioVas-
cular Research Center, Tzu Chi General Hospital, Hualien City, Hualien County,
Taiwan. 3 Department of Medical Biotechnology and Laboratory Sciences,
College of Medicine, Chang Gung University, Taoyuan, Taiwan. 4 Graduate
Institute of Biomedical Sciences Division of Biotechnology, College of Medi-
cine, Chang Gung University, Taoyuan, Taiwan. 5 Hyperbaric Oxygen Medical
Research Lab, Bone and Joint Research Center, Linkou Chang Gung Memorial
Hospital, Taoyuan, Taiwan. 6 Centre for Stem Cell Research, Faculty of Medicine
and Health Sciences, Universiti Tunku Abdul Rahman, Selangor, Malaysia.
7 Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung
University, Taoyuan, Taiwan. 8 School of Medicine, College of Medicine, Chang
Gung University, Taoyuan, Taiwan. 9 Department of Thoracic Medicine, Chang
Gung Memorial Hospital, Taoyuan, Taiwan. 10 Molecular Medicine Research
Center, Chang Gung University, Taoyuan, Taiwan.
Received: 22 October 2024 Accepted: 22 December 2024
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