COL5A1 promotes triple-negative breast cancer progression by activating tumor cell-macrophage crosstalk

doi:10.21203/rs.3.rs-3869020/v1

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COL5A1 promotes triple-negative breast cancer progression by activating tumor cell-macrophage crosstalk

https://doi.org/10.21203/rs.3.rs-3869020/v1

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published 12 Apr, 2024

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Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer. Despite the reported interplay between tumors and tumor-associated macrophages (TAM) in fostering drug resistance and disease progression, the underlying mechanisms governing these interactions remain elusive. Here, it was found an upregulation of Collagen type V alpha 1 (COL5A1) in TNBC tissues, particularly in chemoresistant samples, correlating with an unfavorable prognosis. Functional assays demonstrated that COL5A1 played a pivotal role in fostering cancer growth, metastasis, and doxorubicin (DOX) resistance both in vitro and in vivo. In addition, the cytokine IL-6 produced by COL5A1 overexpressing TNBC cells facilitated M2 macrophage polarization. Reciprocally, TGFβ from M2 macrophages promoted TNBC DOX resistance through mediating the TGFβ/smad3/COL5A1 signaling pathway, therefore constituting a feed-back loop between TNBC cells and macrophages. Mechanistically, COL5A1 was found to interact with TGM2, inhibiting its K48-linked ubiquitination-mediated degradation. This led to an enhanced chemoresistance and increased secretion of IL-6. In summary, these findings unveiled that the upregulation of COL5A1 held significant potential in driving TNBC progression and chemoresistance by modulating macrophage polarization. This supports the proposition that targeting COL5A1 could emerge as an effective strategy against TNBC.

Biological sciences/Cancer/Breast cancer

Biological sciences/Immunology/Tumour immunology

TNBC

COL5A1

chemoresistance

macrophage

Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer-related deaths in women worldwide[1]. Among the various subtypes of breast cancer, triple-negative breast cancer (TNBC) is particularly challenging as it is characterized by the absence of estrogen receptor(ER), progesterone receptor(PR), and human epidermal growth factor receptor 2 (HER2), and representing 15–20% of all breast cancers[2]. The heightened heterogeneity, increased rates of distant metastasis, disease relapse, and lack of validated therapeutic targets pose formidable challenges of TNBC. Presently, chemotherapy serves as the primary therapy for TNBC patients. Nevertheless, a substantial portion of TNBC patients develops drug resistance, rendering treatment ineffective [3]. Consequently, identifying novel therapeutic targets driving TNBC progression and chemoresistance is of utmost importance.

Emerging research has highlighted the extensive interactions between cancer cells and the tumor microenvironment (TME), crucial for sustaining tumor growth, metastasis and drug resistance[4]. The TME in breast cancer is profoundly immunosuppressive and comprises various immune cell populations, including regulatory T cells (Tregs), dendritic cells (DCs), tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and neutrophils[5]. Previous studies have underscored the pivotal role of the TME in maintaining chemoresistance in breast cancer[6]. TAMs, as the predominant infiltrating immune cells in TNBC tissue, exhibit high plasticity, existing in two phenotypic forms depending on the local TME conditions: classically activated macrophages (M1 macrophages), which display proinflammatory and antitumor properties, and alternatively activated macrophages (M2 macrophages), which exhibit anti-inflammatory properties and promote tumor progression. Notably, a heightened prevalence of M2 macrophages in the TME of breast cancer, particularly in malignancy-associated ascites, exhibits a robust correlation with increased tumor growth, invasion, immune evasion, and heightened resistance to chemotherapy[7, 8]. However, the precise roles of TAMs in TNBC progression, particularly in the context of chemoresistance, remain poorly understood. Moreover, the mechanisms underlying how TAMs are educated by the TME to adopt their phenotype and function remain unclear. Therefore, strategies aimed at targeting critical molecules that regulate the interaction between TNBC cells and TAMs, as well as chemoresistance, hold promise as novel therapeutic avenues for preventing and combating chemoresistance.

The findings from the present study have revealed a noteworthy upregulation of Collagen type V alpha 1 (COL5A1) in TNBC tissues, which is strongly associated with an unfavorable prognosis for TNBC patients. Through a series of functional experiments, we successfully validated the critical role of COL5A1 in TNBC progression and chemoresistance. Subsequent investigations further elucidated that the overexpression of COL5A1 prominently induced the polarization of M2-like macrophages with pro-tumor characteristics. This effect was mediated through the activation of the JAK2/STAT3 signaling pathway, facilitated by the heightened secretion of IL-6. Additionally, conditioned macrophages displayed a remarkable capacity to enhance TNBC chemoresistance via the activation of the TGFβ/smad3 pathway. These collective findings highlight the significant involvement of the interplay between breast cancer cells and macrophages as a pivotal mechanism contributing to chemoresistance in TNBC patients. Consequently, the observations provide compelling evidence of the correlation between COL5A1 upregulation and an unfavorable response to chemotherapy in TNBC cases. This deeper understanding of the role of COL5A1 in TNBC pathogenesis and its impact on chemoresistance holds great promise for the identification of potential therapeutic targets to combat these challenges.

Patients and specimens

Tissue samples from breast cancer patients and adjacent normal tissues were collected during surgical procedures at Qilu Hospital of Shandong University. The survival time was defined as the period from the date of surgery to the date of the last follow-up or death. The collection of samples was carried out with informed consent, adhering to the ethical guidelines stipulated by the Shandong University Ethical Committee.

Cell culture and treatment

Three human breast cancer cell lines, namely MDA-MB-231, MDA-MB-468, and HS578T, were cultured routinely in DMEM (Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS) (Cellbox, China) and 1% penicillin/streptomycin. The Thp1 cell line, sourced from the American Type Culture Collection (ATCC, USA), was cultured in RPMI 1640 (Invitrogen, USA) supplemented with 10%FBS (Gibco, USA). These cell lines were authenticated through short tandem repeats (STR) profiling and confirmed to be free of mycoplasma contamination. To induce macrophage adherence, Thp1 cells were treated with 100nmol/L phorbol 12-myristate 13-acetate (PMA) (sigma) for 24h. In some experimental settings, TNBC cells were exposed to doxorubicin or TGFβ inhibitor for 2 days.

For coculture experiments, a total of 5×10⁵ macrophages were seeded in the lower chamber of a 6-well plate, while 5×10⁵ breast cancer cells were added to the upper chamber of a transwell insert with a pore size of 0.4 mm (Millipore). In certain experiments, cocultures were treated with anti-IL6 neutralizing antibodies (Beyotime) according to the manufacturer's instructions. TNBC–conditioned macrophage cells were washed with PBS and replenished with fresh serum-free media. Subsequently, 24h later, conditioned media (CM) were collected and filtered using a 0.22-mm filter.

RNA isolation and qRT-PCR

To extract total RNA from tissues or cells, Trizol reagent (Invitrogen, USA) was used. The concentration of RNA was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher). Subsequently, cDNA synthesis was performed using the PrimeScript reverse transcriptase (RT) reagent kit (TaKaRa, Shiga, Japan). Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was conducted to evaluate the relative mRNA expression. The SYBR green PCR mix (Takara) was used for qRT-PCR, with β-actin serving as the internal control. The primer sequences utilized for qRT-PCR can be found in supplementary Table S1.

Cell proliferation assay and chemosensitivity assay in vitro

The transfected cells were plated in 96-well plates at a concentration of 2000 cells per well. For the chemosensitivity assay, the cell density was adjusted to 5000 cells per well. After an incubation period of overnight, the culture medium was replaced with solutions containing doxorubicin at specified concentrations. At designated time points, 20µl of MTT solution was added to each well and incubated for 4h. Subsequently, the medium was removed, and 100µl of dimethyl sulfoxide (DMSO) was added. The absorbance was measured at 490 nm using a Microplate Reader.

EDU assay

Cell proliferation viability was evaluated using the EdU Proliferation Kit (RiboBio, China). Briefly, 10,000 transfected cells were seeded in a 96-well plate and incubated overnight. After a 2.5h incubation with 50 mM EdU, the cells were fixed using 4% paraformaldehyde (PFA) and stained with Apollo Dye Solution. Nuclei were counterstained with Hoechst dye. Images were captured using an Olympus microscope (Olympus, Tokyo, Japan).

Colony formation assay

After transfection, the breast cancer cells were seeded in 6-well plates at a concentration of 1000 cells per well. Following a 2-week incubation, the cells were washed with PBS, fixed with methanol, and subsequently stained with a 1% crystal violet solution.

Cell cycle and apoptosis assays

Both the cell cycle and apoptosis assays provided insights into cellular processes related to cell division and programmed cell death, respectively. For the cell cycle assay, the transfected cells were stained with propidium iodide (PI) in the dark for 30min. In the cell apoptosis assay, the transfected cells were stained with 5µL of Annexin V-FITC and 5µL of PI, followed by a 15min incubation in the dark. Flow cytometry analysis was then performed to quantify the distribution of cells in different phases of the cell cycle and to determine the percentage of apoptotic cells. The acquired data were analyzed using modfitLT5 and FlowJo software, respectively.

Transwell assays

The cell migration and invasion assays were conducted using a transwell system. A total of 1×10⁵ cells suspended in serum-free medium were added to the upper wells of the transwell inserts, while the lower wells were filled with 700µl of culture medium containing 20% FBS. Following the specified incubation period, the cells that had migrated or invaded through the transwell inserts to the lower surface were quantified.

Wound-healing assay

Cells were seeded and incubated in a 12-well plate until a confluent monolayer was formed. Subsequently, scratches were created on the monolayer using a sterile 10µl pipette tip. The cells were then cultured in serum-free DMEM to eliminate the influence of internal proliferation. Images of the scratched area were captured at the designated time points to monitor cell migration and closure of the scratch.

Western blot

Cellular protein was extracted and separated by 10% SDS-PAGE gels. The separated proteins were then transferred onto 0.22µm PVDF membranes. After blocking with 5% skim milk for 1h, the membranes were incubated overnight at 4°C with specific primary antibodies. Following the primary antibody incubation, the membranes were incubated with secondary antibodies for 2h at room temperature. Protein bands were detected using chemiluminescence, with β-actin serving as a control. The antibodies used in the study are listed in Table S2.

Flow cytometric analysis

To assess the infiltration of macrophages into tumors, tumor tissues were isolated from sacrificed mice. The tissues were finely minced and then digested with collagenase (100 µg/mL) and DNase (0.1 mg/mL) for 2h at 37°C. After digestion, the tissues were filtered through a mesh to obtain cell suspensions. The cell suspensions were treated with RBC lysis buffer to remove red blood cells. Subsequently, the cells were stained with specific antibodies for 30min. The fluorescent dye-conjugated mouse antibodies used for staining included CD11b-FITC (BioLegend), F4/80-PE (BioLegend), CD86-APC (BioLegend), and CD163-APC (BioLegend). In addition, to analyze the phenotypes of PMA-stimulated Thp-1 cells that had been co-cultured with breast cancer cells, the following fluorescent mAbs were used for staining: CD80-PE (BioLegend), CD86-FITC (BioLegend), CD163-APC (BioLegend), and CD206-PE (BioLegend).

Human breast cancer organoids and treatments

Fresh breast cancer tissue specimens were prepared by cutting them into small 1mm³ fragments on ice. The fragments were then digested for 1h at 37°C with gentle shaking. After digestion, the tissues were filtered with a 100µm filter strainer to retain the tissue fragments and centrifuged at 300g for 5min at 4℃. After depleting red blood cells, the cell pellet was resuspended in growth factor-reduced Matrigel (R&D) and seeded into 48-well tissue culture plates with a sufficient Matrigel layer to support the 3D growth of the organoids. The gel was allowed to polymerize for 1h at 37°C and then overlaid with 400µL organoid culture medium. The medium was changed every three days to ensure optimal growth conditions, and the organoids were passaged every one to four weeks.

Co-immunoprecipitation(co-IP)

Co-IP was performed according to the manufacturer's instructions using the specific antibodies and IgG. In brief, cell lysates were incubated with antibodies-conjugated beads at 4℃ overnight. The beads were then extensively washed to remove nonspecific binding, and the immunoprecipitated proteins were eluted by boiling in SDS loading buffer. Western blotting was employed to analyze the immunoprecipitated proteins.

Immunohistochemistry (IHC), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA)

For IHC, tissue sections were deparaffinized and rehydrated, followed by antigen retrieval and blocking of non-specific binding. Then the sections were incubated with primary antibodies (COL5A1,1:500; Ki67,1:1000; TGM2,1:1000; IL-6,1:1000; CD163,1:1000) overnight at 4℃, followed by corresponding secondary antibodies at room temperature for 2h. Subsequently, the tissue slides were stained with diaminobenzidine and counterstained with hematoxylin. An Olympus light microscope was utilized to capture photos of representative areas.

For IF, cells were fixed, permeabilized, and blocked. Then, specific primary antibodies were applied and incubated, followed by the addition of fluorescently labeled secondary antibodies. Finally, the cells were counterstained with DAPI and imaged using an Olympus microscope.

In ELISA experiments, different conditioned breast cancer cells and cocultured macrophages were cultured in 6-well plates, and the supernatant was collected after 48h for quantifying the levels of IL-6, CCL2, CSF1, TGFβ secretion using ELISA kits (RD).

Tumor xenograft model

A total of 1×107 MDA-MB-231 cells, with or without COL5A1 overexpression, were suspended in 200µL of PBS and then subcutaneously injected into the left flanks of 4-6-week-old female BALB/c nude mice. The mice were randomly divided into groups of 5 mice each. The growth of the tumors was monitored weekly by measuring the maximum (L) and minimum (W) lengths of the tumors. After 4 weeks, the mice were euthanized, and the tumors were collected and weighed. The tumor volume was calculated using the formula ½LW2. All animal experiments were conducted following the approved protocols of the Shandong University Animal Care and Use Committee to ensure ethical standards were met.

Statistical analysis

Data analysis was performed using GraphPad Prism 8.0 software. The results were presented as the mean ± standard deviation (SD) from three independent experiments. Student's t-test or ANOVA test was used to compare two groups or more than two groups, respectively, and calculate p-values. COX proportional hazards model multivariate analyses were used to assess the significance of COL5A1 expression and clinicopathological features on overall survival. A p-value less than 0.05 (p < 0.05) was considered statistically significant.

COL5A1 was elevated in breast cancer tissues and associated with an unfavorable prognosis

To identify potential genes indicative of chemoresistance in breast cancer, we conducted an analysis using the TCGA primary TNBC tissue dataset, the GEO TNBC chemotherapy tissue dataset (GSE103668), and TNBC primary and chemoresistant cell lines (231, 231/DOX). Four genes, namely COL5A1, ITGAX, CTSB, and LRRC15, were consistently appeared in all the datasets (Fig. 1A). Given the establish role of COL5A1 in tumor progression and chemoresistance across various tumor types, we chose to focus on its detailed analysis. Analysis indicated that COL5A1 expression was significantly upregulated in TNBC tissues compared to normal tissues, as evidenced by data from TCGA, Metabric, and GEO databases (Fig. 1B, supplementary Fig.S1A). Additionally, TNBC chemoresistance samples exhibited higher COL5A1 expression than chemosensitive samples in GSE103668 (Fig. 1B). qRT-PCR analysis revealed elevated COL5A1 mRNA expression levels in TNBC tissues compared to non-tumor tissues (Fig. 1C). Moreover, when compared to paired normal counterparts, TNBC tissues exhibited upregulated mRNA expression of COL5A1 (Fig. 1D). Immunohistochemistry (IHC) evaluation of COL5A1 protein expression in TNBC tissues showed significantly higher levels compared to non-tumor tissues (Fig. 1E). Furthermore, COL5A1 expression was found to be higher in chemoresistant TNBC tissues than in chemosensitive TNBC tissues (Fig. 1F). To assess the clinical significance of COL5A1 expression, we analyzed 76 TNBC patients and considered various clinicopathological characteristics (supplementary Table.S3). Results indicated a significant association between COL5A1 expression and distant metastasis, although no significant associations were observed with age, tumor size, histologic grade, lymph node (LN) metastasis, and Ki67 expression. Univariate and multivariate Cox regression analyses identified LN metastasis and COL5A1 expression as independent prognostic indicators for the overall survival of TNBC patients(supplementary Table.S4). Furthermore, we compared the expression levels of COL5A1 in TNBC cells (MDA-MB-231) and normal cells (MCF-10A). We found that COL5A1 expression was significantly higher in TNBC cells, and within the TNBC cell line, the DOX-resistant MDA-MB-231/DOX cells exhibited even higher expression levels compared to the parental MDA-MB-231 cells (Fig. 1G,H). The immunofluorescent staining further confirmed this result (Fig. 1I). Kaplan-Meier analysis illustrated that TNBC patients with high COL5A1 expression had poorer overall survival and disease-free survival (Fig. 1J, supplementary Fig.S1B). In conclusion, our study revealed a strong association between elevated COL5A1 expression and poor prognosis in TNBC patients, suggesting its potential as a biomarker for chemoresistance in this aggressive breast cancer subtype.

COL5A1 promoted the progression of TNBC cells in vitro

We investigated the functional role of COL5A1 in the tumorigenesis and progression in TNBC cells, including MDA-MB-231 and MDA-MB-468. Through MTT, EdU, and clony formation assays, we observed an increase in cell proliferation upon upregulation of COL5A1 (Fig. 2A-C). Flow cytometry analysis was then conducted to examine the effect of COL5A1 on the cell cycle profile and apoptosis. Overexpression of COL5A1 promoted cell cycle progression, as evidenced by a decrease in the number of cells in the G1 phase and an increase in the S phase population (Fig. 2D). Additionally, COL5A1 overexpression resulted in a reduced apoptotic rate (Fig. 2E). Western blot analysis consistently indicated that COL5A1 upregulation elevated the protein levels of cell cycle-related proteins and suppressed apoptosis (Fig. 2F). Migration and invasion abilities were assessed using transwell assays, which demonstrated a significant enhancement in these properties in breast cancer cells with COL5A1 overexpression, consistent with the results of the wound healing assay (Fig. 2G-I). Epithelial-mesenchymal transition (EMT) is a critical process associated with cancer metastasis. Western blot analysis revealed that COL5A1 overexpression led to the downregulation of epithelial markers while concurrently upregulating mesenchymal markers, indicating activation of EMT (Fig. 2J). Additionally, COL5A1 knockdown experiments revealed inhibited cell growth, migration, and invasion, coupled with enhanced cell apoptosis (supplementary Fig.S2). Collectively, our findings strongly supported the notion that COL5A1 functions as a tumor promoter in TNBC cells by promoting cell proliferation, inducing cell cycle progression, suppressing apoptosis, enhancing migration and invasion capabilities, and driving EMT.

COL5A1 promoted the doxorubicin resistance of breast cancer cells in vitro

We aimed to explore the role of COL5A1 in conferring resistance to doxorubicin treatment in TNBC cells. Firstly, we identified that doxorubicin-resistant breast cancer cells(231/DOX) had the higher mRNA expression levels of COL5A1 than parental cells(231)(Fig. 3A), suggesting its involvement in promoting doxorubicin resistance. Notably, low concentrations of DOX were found to promote COL5A1 expression, while high concentrations inhibited its expression (supplementary Fig.S3A). The IC50 value of doxorubicin was obviously higher in breast cancer cells overexpressing COL5A1 compared to control cells, indicating enhanced resistance (Fig. 3B, supplementary Fig.S3B). Consistent with these findings, 231/DOX cells transfected with si-COL5A1 exhibited attenuated cell proliferation and decreased resistance to DOX in a dose-dependent manner (Fig. 3C,D, supplementary Fig.S3C). COL5A1 overexpression suppressed DOX-induced apoptosis, while COL5A1 knockdown led to enhanced apoptosis (Fig. 3E,F, supplementary Fig.S3D,E), as confirmed by flow cytometry and western blot assays. Furthermore, we constructed a patient-derived organoid (PDO) model to investigate the correlation between COL5A1 expression and PDO sensitivity to DOX. The results indicated that a higher expression level of COL5A1 tended to promote proliferation and increase sensitivity to DOX in PDOs (Fig. 3G). These observations underscored the significant role of COL5A1 in doxorubicin resistance in TNBC cells.

COL5A1 induced macrophage polarization toward an M2 phenotype through IL-6/JAK2/STAT3 signaling pathway

To investigate the potential regulatory role of COL5A1 in the interaction between cancer cells and infiltrating immune cells in tumor tissues, we utilized CIBERSORT algorithm based on GEO dataset. Intriguingly, among the various infiltrating immune cells, macrophages, especially the M2-type, showed a strong and remarkable positive correlation with COL5A1 expression (Fig. 4A). Additionally, chemoresistant TNBC patients exhibited a higher proportion of M2-type macrophages and a lower proportion of M1-type macrophages compared to chemosensitive patients, as observed in GEO data (Fig. 4B). IHC analysis further verified higher CD163-positive macrophage infiltration in chemoresistant TNBC tissues (Fig. 4C). Consistent with this, qRT-PCR and flow cytometry analysis confirmed that 231/DOX had a stronger ability to induce macrophage polarization toward M2-type in vitro (supplementary Fig.S4A,B). Spearman Correlation analysis revealed a positive correlation between COL5A1 and CD163 expression (Fig. 4D). To validate the impact of COL5A1 on macrophage polarization, we utilized Thp-1 cells, which can differentiate into macrophage-like cells, and co-cultured them with COL5A1-overexpressing TNBC cells in a transwell system. The expression of macrophage markers (CD80, CD86, CD163, CD206) and macrophage cytokines (IL-1, CCL2, IL-10, TGFβ, VEGF) were assess by qRT-PCR. Compared with vector control cells, COL5A1-overexpressing TNBC cells significantly increased CD163, CD206, IL-10, TGFβ, VEGF expression indicative of M2 macrophages and markedly decreased CD80, CD86, IL-1, CCL2 expression indicative of M1 macrophages in macrophages (Fig. 4E, supplementary Fig.S4C). Flow cytometry analysis further supported these findings, with higher percentages of CD163- and CD206-positive cells and lower percentages of CD80- and CD86-positive cells observed in Thp-1 cells cocultured with COL5A1-overexpressing TNBC cells compared to those co-cultured with vector control cells (Fig. 4F). Additionally, both qRT-PCR and flow cytometry consistently demonstrated a reduction in the percentage of M2 macrophages, coupled with an increase in M1 macrophages in Thp-1 cells cocultured with COL5A1 knockdown TNBC cells compared to those from control cells (supplementary Fig.S4D-F). As the IL-6/JAK/STAT3 and PI3K/AKT/mTOR pathway plays crucial roles in promoting M2 polarization[8, 9], our investigation revealed that stimulation of Thp-1 cells with COL5A1-overexpressing TNBC cells led to an increased expression of p-JAK2 and p-STAT3, while the expression of p-AKT and p-MTOR remained unchanged (Fig. 4G). Furthermore, the expression of p-JAK2 and p-STAT3 was downregulated in Thp-1 cells stimulated by COL5A1 knockdown TNBC cells (supplementary Fig.S4G).

Subsequently, we examined the inflammatory factors associated with macrophage polarization in the conditioned medium of COL5A1-overexpressing TNBC cells and control cells. qRT-PCR demonstrated significant alterations in IL-6 and CSF-1 in COL5A1-overexpressing TNBC cells compared to control cells (supplementary Fig.S4H). Of these inflammatory factors, IL-6 stood out as the most notably upregulated and abundant cytokine. Both ELISA and Western blot further confirmed a significant elevation or reduction in IL-6 in COL5A1-overexpressing or knockdown TNBC cells, respectively (Fig. 4H,I, supplementary Fig.S4I,J). These results were consistent with the notion that macrophage polarization was activated by the JAK2/STAT3 pathway. To delve deeper into whether COL5A1-overexpressing TNBC cells-conditioned macrophages polarize through activating the IL-6/JAK2/STAT3 pathway, we assessed macrophage markers. The results demonstrated that COL5A1-overexpressing TNBC cells exhibited a diminished ability to polarize M2-type macrophages after coculturing with IL-6 neutralizing antibodies or JAK2 inhibitor-pretreated COL5A1-overexpressing TNBC cells (Fig. 4J,K, supplementary Fig.S4K). Mechanistically, treatment with IL-6 neutralizing antibodies or JAK2 inhibitor inhibited the JAK2/STAT3 pathway (Fig. 4L). These collective observations imply that COL5A1 might contribute to the polarization of M2 macrophages in the progression of TNBC.

COL5A1 targeted TGM2 through suppressing TGM2 K48-ubiquitination to inhibit the proteasomal degradation.

To elucidate the intracellular targets of COL5A1, we employed MDA-MB-468 cells to conduct a mass spectrometry-based formaldehyde crosslinking assay, identifying TGM2 as a putative binding partner of COL5A1 (Fig. 5A,B). The interaction between COL5A1 and TGM2 was further confirmed in 293T cells (Fig. 5C). Endogenous interactions between COL5A1 and TGM2 were additionally detected within MDA-MB-231 and MDA-MB-468 cells (Fig. 5D), and the two proteins colocalized in these cells (Fig. 5E, supplementary Fig.S5A). Interestingly, we found that changes in COL5A1 expression did not affect the mRNA levels of TGM2, as determined by qRT-PCR (Fig. 5F, supplementary Fig.S5B). However, western blot analysis provided positive results. Overexpression of COL5A1 upregulated the expression of TGM2, while knockdown of COL5A1 downregulated the expression of TGM2 (Fig. 5G, supplementary Fig.S5C). This phenomenon suggested that COL5A1 might be involved in the post-translational modification process but had no effect on mRNA transcription. Moreover, CHX chase assays demonstrated that overexpression of COL5A1 in MDA-MB-231 and MDA-MB-468 cells extended the half-life of the TGM2 protein (Fig. 5H), while knockdown of COL5A1 promoted TGM2 protein degradation in MDA-MB-231 and HS578T cells (supplementary Fig.S5D). The proteasome inhibitor MG132 was sufficient to reverse the knockdown COL5A1-induced TGM2 degradation, whereas chloroquine (CQ) had no impact on such degradation, inhibiting lysosome activity (Fig. 5I). Ubiquitination is a post-translational modification that enables proteins to be degraded by the ubiquitin-proteasome degradation pathway[10]. Therefore, we evaluated whether COL5A1 could induce TGM2 ubiquitination. The results showed that COL5A1 decreased the ubiquitinated levels of TGM2(Fig. 5J). To better classify the COL5A1-induced TGM2 ubiquitin chain linkage on TGM2, we used two commonly ubiquitin mutant isoforms K48 and K63 for experiment. The result showed that COL5A1 markedly decreased the K48-linked ubiquitination of TGM2 but had no impact on K63-linked ubiquitination (Fig. 5K). Together, these results suggest that COL5A1 regulated the expression of TGM2 through inhibiting K48-ubiquitination-mediated degradation.

TGM2 knockdown partially reversed the promotion effect of COL5A1 overexpression in TNBC cells

We further investigated whether the knockdown of TGM2 could attenuate the promotional effects caused by the overexpression of COL5A1. The result demonstrated that TGM2 knockdown suppressed the proliferative, migratory, and invasive effects, as well as DOX resistance promoted by COL5A1 overexpression in breast cancer cells (Fig. 6A-D, supplementary Fig.S6A-C). As TGM2 had been known to promote IL-6 secretion through the NF-κB pathway[11], we further examined whether COL5A1 regulated IL-6 expression through TGM2. ELISA and qRT-PCR results demonstrated that the knockdown of TGM2 inhibited the pro-secretion of IL-6 in COL5A1 overexpression cells (Fig. 6E, supplementary Fig.S6D). Western blot further confirmed that TGM2 regulated COL5A1-induced IL-6 secretion through the PI3K/AKT/NF-κB pathway (Fig. 6F). Furthermore, we investigated the impact of TGM2 knockdown on macrophage polarization in Thp-1 cells co-cultured with COL5A1 overexpression cells. Simultaneous knockdown of TGM2 restored the downregulation of M1 markers and the upregulation of M2 markers induced by COL5A1 overexpression (Fig. 6G,H, supplementary Fig.S6E). These findings indicated that the interaction between COL5A1 and TGM2 played a crucial role in mediating the promotion of chemoresistance and polarization of M2 macrophages in TNBC cells.

COL5A1 overexpression TNBC-conditioned macrophage remarkably promoted the chemoresistance process through TGFβ in TNBC cells

To evaluate the functional impact of M2-type macrophages on TNBC cells and their role in promoting chemoresistance, we employed a double-conditioned medium (DCM) treatment strategy (Fig. 7A). Thp-1 was differentiated into macrophages and co-cultured with TNBC cells transfected with COL5A1-vector for 48h. Subsequently, fresh medium was added and incubated for an additional 24h to generate the DCM. This DCM was then used to treat parental TNBC cells with DOX in subsequent in vitro assays. Compared to control TNBC cells, the IC50 value of DOX significantly increased in DCM-treated parental TNBC cells overexpressing COL5A1 (Fig. 7B). Furthermore, in parental TNBC cells subjected to DOX treatment, exposure to DCM derived from TNBC cells with COL5A1 overexpression resulted in a substantial reduction in DOX-induced apoptotic death and enhanced migration compared to DCM from control cells (Fig. 7C,D, supplementary Fig.S7A,B). Notably, a positive correlation was observed between the expression levels of COL5A1 and those of TGFβ and IL-10 (p<0.0001,respectively), while no correlation was found with the expression of VEGFA, all of which were primary secreted cytokines associated with M2-type macrophages (Fig. 7E, supplementary Fig.S7C). Subsequent ELISA confirmed a significant increase in TGFβ levels in M2-type macrophages cocultured with COL5A1-overexpressing TNBC cells (Fig. 7F),while knockdown of COL5A1 had the opposite effect (supplementary Fig.S7D). Therefore, TGFβ was selected for further analysis.

To assess the necessity of TGFβ in COL5A1-mediated induction of M2-type macrophages and DOX resistance, we introduced a TGFβ inhibitor into the coculture system. Remarkably, the addition of the TGFβ inhibitor significantly increased the number of apoptotic cells treated with DOX (Fig. 7G). Furthermore, we added exogenous recombinant TGFβ to the culture medium of TNBC cells transfected with siCOL5A1. The results showed that TGFβ dose-dependently reduced drug-induced apoptosis and reversed the effect of COL5A1 knockdown, leading to a decrease in DOX-induced apoptosis (Fig. 7H supplementary Fig.S7E,F). Given TGFβ's role in wound healing by regulating collagen synthesis[12], we investigated whether TGFβ derived from DCM regulated COL5A1 expression. Indeed, parental TNBC cells treated with COL5A1-overexpressing TNBC cell DCM exhibited higher mRNA and protein expression of COL5A1 compared to incubation in control cell-derived DCM. Notably, the TGFβ inhibitor could suppress this effect (Fig. 7I,J). Moreover, TGFβ significantly increased COL5A1 protein levels in a dose-dependent manner (supplementary Fig.S7G). To confirm that DCM derived TGFβ induced COL5A1 expression through SAMD complex (a key regulator of TGFβ signaling), we assessed the expression of SMAD2/3 in DCM-treated parental TNBC cells. The findings revealed an upregulation of SMAD3 in parental TNBC cells treated with COL5A1-overexpressing TNBC cell DCM, which was subsequently downregulated upon the addition of the TGFβ inhibitor (Fig. 7J). Overexpression of SMAD3 promoted the expression of COL5A1, while knockdown of SMAD3 had the opposite effect (Fig. 7K, supplementary Fig.S7H). These findings suggested that TGFβ derived from TAMs induced chemoresistance in TNBC cells, establishing a positive feedback loop that activated COL5A1 and TGFβ in TNBC.

Enforced expression of COL5A1 promoted the growth, doxorubicin resistance, and M2 macrophages polarization of breast cancer in vivo

To elucidate the functional significance of COL5A1 as a regulator of doxorubicin resistance, we employed COL5A1-specific shRNAs and an overexpression plasmid to efficiently knockdown and overexpress COL5A1. Subsequently, the cells were inoculated into the flanks of BALB/c nude mice. Upon reaching a tumor volume of 50 mm^3, the mice received intravenous injections of either PBS or DOX. Consistent with our in vitro results, Overexpression of COL5A1 resulted in an augmentation of both tumor volume and weight. Notably, in mice injected with DOX, the tumors exhibited reduced size and weight compared to those in mice treated with PBS, underscoring the inhibitory impact of DOX on TNBC growth in vivo (Fig. 8A-C). Notably, tumors in mice injected with DOX, where COL5A1 was overexpressed, displayed significantly larger sizes and greater weights in comparison to tumors in DOX-injected mice with control cell implants (Fig. 8A-C). This implies that the overexpression of COL5A1 diminished the anti-tumor efficacy of DOX. The loss of COL5A1 in TNBC cells was associated with a decrease in tumor volume relative to controls, while DOX treatment further exaggerated this difference, resulting in a decrease in tumor volume (Fig. 8E-G). IHC analysis revealed that alterations in the expression levels of COL5A1, TGM2, and IL-6 in the tumor tissues corresponded with the in vitro results (Fig. 8D). The number of Ki67⁺ tumor cells was notably decreased in DOX group, and this decrease was further enhanced by COL5A1 upregulation (Fig. 8D). Notably, CD163 staining indicated a robust increase in the number of infiltrated M2 macrophages (CD163⁺ macrophages) in TNBC tissues with high COL5A1 expression (Fig. 8D). Furthermore, correlation analysis revealed a positive association between the response to DOX and high COL5A1 expression with CD163 + macrophage infiltration in TNBC (Fig. 8D). Additionally, a substantial decrease in the expression of infiltrated CD163⁺ macrophages was observed in the tumors of mice treated with DOX (Fig. 8D). To further assess the impact of COL5A1 on macrophage infiltration, flow cytometry analysis was performed. The COL5A1 overexpression group exhibited a significant increase in the number of M2 macrophages, accompanied by a decrease in the number of M1 macrophages (Fig. 8I). Meanwhile, treatment with DOX resulted in a decrease in both M1 and M2 macrophage numbers (Fig. 8I). COL5A1 downregulation yielded the opposite phenotypes (Fig. 8H, J). These results demonstrated that COL5A1 amplified breast cancer resistance to DOX and correlated positively with the polarization of M2 macrophages.

Together, our study revealed that COL5A1 overexpression in TNBC enhanced tumor progression, chemoresistance and promoted the polarization of M2 type macrophage by regulating the IL-6/JAK2/STAT3 pathway through decreasing the K48-linked ubiquitination of TGM2. Reciprocally, the elevated expression of TGFβ by COL5A1-overexpressing TNBC-educated macrophages significantly promoted the expression of COL5A1 and chemoresistance of TNBC cells via the TGFβ/smad3 axis in a feedback loop (Fig. 8K).

TNBC is characterized by its aggressive nature and resistance to chemotherapy, contributing significantly to disease progression, relapse, and metastasis, ultimately accounting for over 90% of deaths in TNBC patients[13, 14]. Unveiling novel targets is imperative for understanding the underlying mechanisms and devising strategies to overcome chemoresistance, ultimately enhancing the prognosis of TNBC patients. This study represents the first demonstration that COL5A1 can modulate the TME and significantly contribute to chemoresistance in TNBC. Notably, COL5A1 overexpression not only directly promoted chemoresistance in TNBC cells but also induced the polarization of macrophages toward the M2 type by upregulating IL-6 through the JAK2/STAT3 signaling pathway. In a reciprocal manner, these conditioned macrophages played a pivotal role in enhancing TNBC chemoresistance via the TGFβ/smad2/3 pathway. Mechanistically, COL5A1 interacted with TGM2, inhibiting its degradation through K48-linked ubiquitination. Knockdown of TGM2 compromised the functionality of COL5A1 in TNBC. Overall, our findings offered both mechanistic insights and translational implications into how collagen-mediated modulation of the TME promotes chemoresistance in TNBC.

COL5A1 is a member of the collagen family located on the long arm of chromosome 9 and expressed in both tendons and ligaments[15]. Despite its structural role being relatively minor in the collagen hierarchy, COL5A1 plays a functionally significant role, particularly in regulating fiber diameter and the assembly of collagen fibers. Recent research underscores the varied biological roles played by COL5A1, including involvement in cell migration, differentiation, embryonic development, and the preservation of cellular stemness. Although investigations into the expression and function of COL5A1 in human malignancies are relatively sparse, elevated levels of COL5A1 have been identified in conditions such as adrenocortical carcinoma, clear cell renal cell carcinoma, bladder urothelial carcinoma, gastric cancer, and lower-grade glioma, demonstrating an association with unfavorable survival outcomes[16–18]. Furthermore, COL5A1 overexpression has been implicated in enhancing ovarian cancer cell progression and metastasis, with a correlation noted with tumor-infiltrating immune cells[19]. Nevertheless, the impact of COL5A1 on tumor progression and chemoresistance, along with the underlying mechanisms in TNBC, remains insufficiently explored. This present study addressed this gap by identifying elevated COL5A1 expression in TNBC tissues, chemoresistant TNBC cells, and chemoresistant TNBC tissues. This observation was substantiated by data from multiple public datasets and RNA-seq analyses, with validation from samples in our own cohort. Furthermore, our findings established a correlation between high COL5A1 expression and unfavorable prognostic outcomes in TNBC patients. Subsequent investigations underscored the functional role of COL5A1 in promoting TNBC cell proliferation, migration, invasion, and doxorubicin resistance.

Macrophages play a pivotal role in the TME. TAMs are derived mainly from circulating Ly6Chi monocytes, which promote tumor progression [20]. Increased levels of TAMs have been correlated with unfavorable survival outcomes in a variety of cancer types, including melanoma, colorectal cancer, bladder cancer, and lung cancer[21–24]. The classical proinflammatory cytokine IL-6 is known to facilitate the transition of TAMs toward the M2-type, often regarded as a Th2 cytokine[25, 26]. IL-6 activates JAK and STAT3, inducing anti-inflammatory effects that promote M2 macrophage polarization[8]. Our study established a significant and positive correlation between COL5A1 expression and the immune component, particularly M2-type macrophages, in the TME of chemoresistant TNBC tissues, as evidenced by GEO datasets. This correlation was further validated through in vitro co-culture experiments simulating COL5A1 overexpression TNBC conditions. Notably, major M2 macrophage-related markers CD163 and CD206 were markedly upregulated in macrophages conditioned by COL5A1 overexpression TNBC. Additionally, an increased proportion of F4/80⁺ CD163⁺macrophages in COL5A1 overexpression TNBC tissues compared to control TNBC tissues underscored the significant role of COL5A1 in skewing macrophages towards a pro-tumor (M2) phenotype. To elucidate the underlying mechanism, we screened the cytokine profile of COL5A1 overexpression TNBC cells and identified elevated secretion of IL-6 compared to control cells. Subsequent experiments using neutralizing antibodies confirmed IL-6 as a key factor mediating the macrophage phenotype switch induced by COL5A1 overexpression TNBC cells. Furthermore, we observed increased activation of JAK2 and STAT3 in macrophages co-cultured with COL5A1 overexpression TNBC cells. Crucially, the administration of an IL-6-specific neutralizing antibody partially alleviated this effect, suggesting that COL5A1 overexpression in TNBC cells induced the formation of M2 subtype TAMs by releasing IL-6 and modulating the JAK2/STAT3 signaling pathway.

Recent insights emphasize the intricate interplay between cancer cells and infiltrating immune cells in the context of chemotherapy response[27]. This reciprocal interaction involves direct contacts and the release of cytokines, influencing the overall response to treatment and opening avenues for innovative oncotherapeutic strategies. Notably, M2 macrophages have been identified as key contributors to chemoresistance by releasing chemoprotective factors such as lysosomal enzymes, cathepsins B and S, shielding tumor cells from direct cytotoxic effects[28]. Additionally, the release of MFG-E8 by M2-like macrophages activates the STAT3 signaling pathway associated with cancer stem cells, thereby enhancing chemoresistance in colon cancer[29]. Herein, we observed a striking capacity of COL5A1-overexpressing TNBC cells to educate macrophages, resulting in enhanced DOX resistance compared to control TNBC cells. This effect was particularly potent, underscoring the crucial role of COL5A1-overexpressing TNBC-educated TAMs in driving TNBC chemoresistance. These observations prompted a deeper investigation into the underlying mechanisms. TAMs are known to secrete various cytokines, among which TGFβ is commonly associated with promoting tumor chemoresistance[30, 31]. Consistent with this, our study revealed that TGFβ derived from TAMs induced chemoresistance in TNBC cells, establishing a feedback loop between TNBC cells and macrophages. The existence of this feedback loop highlights the importance of targeting both the tumor cells and the tumor microenvironment to overcome chemoresistance in TNBC. By disrupting this reciprocal interaction, it may be possible to enhance the efficacy of chemotherapy and prevent tumor progression.

TGM2, a versatile enzyme involved in extracellular matrix degradation, apoptosis, chemoresistance, and signal transduction[32, 33]. Aberrant TGM2 expression has been validated in glioma, colon cancer, pancreatic cancer, ovarian cancer, and other malignancies and is associated with poor chemo-response and patient survival[34–37]. Moreover, TGM2 has been found to regulate the secretion of pro-inflammatory cytokines in different cancer types[38]. This study pioneered the revelation that COL5A1 contributed to the maintenance of TGM2 stability by impeding its ubiquitination and subsequent degradation. Intriguingly, our exploration indicated that COL5A1 exerted a regulatory effect on the K48-linked ubiquitination of TGM2, while leaving its K63-linked ubiquitination unaffected. Importantly, our findings demonstrated that the suppression of TGM2, achieved through knockdown, could mitigate the progression, chemoresistance, and macrophage polarization induced by COL5A1 in TNBC cells. This newfound understanding may pave the way for the development of targeted therapies aimed at disrupting the COL5A1-TGM2 axis and improving clinical outcomes for TNBC patients.

In summary, our study unveils the multifaceted role of COL5A1 in TNBC. We have demonstrated that COL5A1 plays a multi-faceted role in promoting TNBC chemoresistance and shaping the tumor microenvironment. By enhancing the polarization of macrophages towards a pro-tumor M2 phenotype through IL6 secretion and JAK2/STAT3 signaling, COL5A1 contributes to the formation of an immunosuppressive TME. Reciprocally, COL5A1-overexpressing TNBC-educated macrophages significantly contribute to TNBC chemoresistance by upregulating COL5A1 through the activation of the TGFβ/smad2/3 pathway. Additionally, our findings highlight the role of TGM2, regulated by COL5A1, in suppressing K48-linked ubiquitination and proteasomal degradation, thereby promoting these observed phenomena. Targeting COL5A1, TGM2, and the macrophage polarization pathways could prove effective in overcoming chemoresistance and improving patient outcomes. Overall, our study underscores the significance of the COL5A1-mediated pathways in TNBC and opens up new avenues for future research and the development of targeted therapies in this aggressive breast cancer subtype.

Competing interests

The authors declare no potential competing interests.

Author Contributions

QFY and XC designed the experiment; XC, CAM, YML, YRL, TC and DL performed the experiments; XC and CAM performed data analysis and prepared the figures/tables; DL, WJZ, LJW collected samples; XC, CAM and DWH wrote the paper; XC, NZ and QFY revised the paper.

Funding

This work was supported by Special Foundation for Taishan Scholars (No. ts20190971), Foundation from Clinical Research Center of Shandong University (No.2020SDUCRCA015), National Natural Science Foundation of China (No.81902697, No.82373267, and No.81972475).

Acknowledgement

We thank TCGA,GEO and Metabric databases for providing data.

Data availability

Source data and reagents are available from the corresponding author upon reasonable request.

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COL5A1 promotes triple-negative breast cancer progression by activating tumor cell-macrophage crosstalk

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Abstract

Figures

Introduction

Materials and methods

Patients and specimens

Cell culture and treatment

RNA isolation and qRT-PCR

Cell proliferation assay and chemosensitivity assay in vitro

EDU assay

Colony formation assay

Cell cycle and apoptosis assays

Transwell assays

Wound-healing assay

Western blot

Flow cytometric analysis

Human breast cancer organoids and treatments

Co-immunoprecipitation(co-IP)

Immunohistochemistry (IHC), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA)

Tumor xenograft model

Statistical analysis

Results

COL5A1 was elevated in breast cancer tissues and associated with an unfavorable prognosis

COL5A1 promoted the progression of TNBC cells in vitro

COL5A1 promoted the doxorubicin resistance of breast cancer cells in vitro

COL5A1 induced macrophage polarization toward an M2 phenotype through IL-6/JAK2/STAT3 signaling pathway

TGM2 knockdown partially reversed the promotion effect of COL5A1 overexpression in TNBC cells

COL5A1 overexpression TNBC-conditioned macrophage remarkably promoted the chemoresistance process through TGFβ in TNBC cells

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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