Breast cancer cell-derived adrenomedullin confers cancer-associated adipose remodeling through the cAMP/Creb1/Zeb1 axis.

Upon penetrating the basement membrane, breast cancer cells directly interact with their surrounding adipose tissue, which forms a unique tumor-associated adipose microenvironment (TAME). However, the underlying mechanism of lipid metabolic remodeling in the TAME remains elusive. Herein, we report a Zeb1-orchestrated bidirectional communication between breast cancer cells and their adjacent cancer-associated adipocytes (CAAs). At the molecular level, breast cancer cells, through the secretion of adrenomedullin (AM), induce downregulation of Zeb1 expression to activate the Atgl/Hsl/Scd-dependent lipolysis in CAAs, resulting in the release of palmitoleic acid (POA) into the TAME. In turn, the increased POA in breast cancer competes with arachidonic acid (ARA) for the phospholipid synthesis, leaving more ARA is utilized for PDG2 production to trigger the malignant progression of breast cancer and AM production. Importantly, disruption of Zeb1-dependent lipolytic activity and/or membrane phospholipid remodeling within the TAME dramatically diminishes the aggressiveness of breast cancer in vitro and in vivo.

ErbB2 is overexpressed in 15-20% of breast cancer, which is associated with malignancy and poor prognosis. We previously reported that ErbB2 supports malignant progression of breast cancer by upregulating lactate dehydrogenase A (LDHA), an important enzyme in glycolysis. However, whether ErbB2 promotes breast cancer progression through other glycolytic enzymes remains unclear. Hexokinase 1 (HK1) and hexokinase 2 (HK2) are the first rate-limiting enzymes of glycolysis and both of them are increased in breast cancer. Here, we aim to investigate whether ErbB2 upregulates HK1 and HK2 and the role of HK1 and HK2 in the malignant progression of ErbB2-overexpressing breast cancer. In current study, we found that the mRNA level of ErbB2 was positively correlated with that of HK1 and HK2, respectively. Moreover, ErbB2 upregulated the protein levels of HK1 and HK2 in breast cancer cells. We also found that both siHK1 and siHK2 significantly inhibited the proliferation, migration and invasion of ErbB2-overexpressing breast cancer cells. Taken together, our findings suggested that ErbB2 promoted the malignant progression of breast cancer cells by upregulating HK1 and HK2, and HK1 and HK2 might serve as promising therapeutic targets for ErbB2-overexpressing breast cancer.

Recent studies have demonstrated that cancer-associated adipocytes (CAAs) in the tumor microenvironment are involved in the malignant progression of breast cancer. However, the underlying mechanism of CAA formation and its effects on the development of breast cancer are still unknown. Here, we show that CSF2 is highly expressed in both CAAs and breast cancer cells. CSF2 promotes inflammatory phenotypic changes of adipocytes through the Stat3 signaling pathway, leading to the secretion of multiple cytokines and proteases, particularly C-X-C motif chemokine ligand 3 (CXCL3). Adipocyte-derived CXCL3 binds to its specific receptor CXCR2 on breast cancer cells and activates the FAK pathway, enhancing the mesenchymal phenotype, migration, and invasion of breast cancer cells. In addition, a combination treatment targeting CSF2 and CXCR2 shows a synergistic inhibitory effect on adipocyte-induced lung metastasis of mouse 4T1 cells in vivo. These findings elucidate a novel mechanism of breast cancer metastasis and provide a potential therapeutic strategy for breast cancer metastasis.

Background: Some HER2-positive (HER2+) breast cancer patient are resistant to anti-HER2 therapy, and the potential reasons of anti-HER2 drug resistance remain unclear. It has been demonstrated that circRNAs play an important role in breast cancer progression, however, the role of circRNAs in anti-Her2 drug resistance remains unknown. In our study, we found and identified a HER2-related circRNA that is associated with malignant progression of HER2 positive breast cancer and preliminary explore their potential mechanisms and clinical significance. Materials and Methods: To identify the HER2-related circRNAs, the deep circRNAs RNA sequencing was performed between the HER2 positive and HER2 negative human breast cancer tissues, and then selected the candidate circRNAs using qPCR to verify the expression level. K-M model was used to define the prognostic performance of circRNA candidates in a breast cancer patient cohort with anti-HER2 drug treatment(N=120). Divergent primers assay and actinomycin D experiments were used to identify the circular features of circRNA candidates. To detect the biological function in breast cancer cells, we knocked-down or over-expressed the circular RNA by siRNA and plasmid transfection. We conducted CHiRP (Chromatin Isolation by RNA Purification) assay to pull down protein complex using biotinylated circRNA probe, and performed mass spectrometric to find out the potential binding protein. Western blot and immunofluorescence (IF) verified that circRNA interacted the protein candidate Results: In this study, we identified a HER2 -Associated CircRNA (named HEACA), and found that divergent primers were amplified in cDNA but not gDNA. Convergent primers amplified in the both cDNA and gDNA. As expected, HEACA was more stable than its linear mRNA when both of them were treated by actinomycin D. Clinical data indicated that HER2+ breast cancer patients with high expression level of HEACA was significantly associated with larger tumour size, more much lymphatic metastasis and higher ki67 index. Kaplan-Meier survival curves showed that HER2+ breast cancer patients with high HEACA who received anti-HER2 therapy has shorter disease-free survival (DFS) and overall survival (OS) than those with low HEACA. Next, we found that know-down HEACA significantly inhibited the proliferation of HER2 positive breast cancer cells through cell variability assay and colony formation, while over-express HEACA highly increased cell proliferation. However, neither know-down nor over-express resulted in affecting the biological function in MCF-7 cells. CircRNA pull down assay and mass spectrometric indicated HEACA may bind to Growth factor receptor-bound protein 7(GRB7). Western blot and IF showed that the protein expression of GRB7 increased or decreased by knocking down or over-express HEACA. Furthermore, we demonstrated that upregulated HEACA promoted breast cancer progression via GRB7/AKT axis in HER2+ cell lines, consequently resulted in a malignant progression of breast cancer cells in vitro and in vivo. Conclusions: HEACA-GRB7/AKT axis plays a critical role in HER2+ breast cancer progression and may be a predictive biomarker for anti-her2 therapy for HER2+ BC patients. Citation Format: Yun Ling, Liang Gehao, Zihao Liu, Zhenluan Tian, Wenjing Zhong, Wanyi Lin, Erwei Song, Gong Chang. circHEACA promotes anti-HER2 drug resistance by HEACA-GRB7/AKT axis in HER2+ breast cancer patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-04-05.

BackgroundBreast cancer cells recruit surrounding stromal cells, such as cancer-associated fibroblasts (CAFs), to remodel collagen and promote tumor metastasis. Adipocytes are the most abundant stromal partners in breast tissue, local invasion of breast cancer leads to the proximity of cancer cells and adipocytes, which respond to generate cancer-associated adipocytes (CAAs). These cells exhibit enhanced secretion of extracellular matrix related proteins, including collagens. However, the role of adipocyte-derived collagen on breast cancer progression still remains unclear.MethodsAdipocytes were cocultured with breast cancer cells for 3D collagen invasion and collagen organization exploration. Breast cancer cells and adipose tissue co- implanted mouse model, clinical breast cancer samples analysis were used to study the crosstalk between adipose and breast cancer cells in vivo. A combination of proteomics, enzyme-linked immunosorbent assay, loss of function assay, qPCR, western blot, database analysis and chromatin immunoprecipitation assays were performed to study the mechanism mediated the activation of PLOD2 in adipocytes.ResultsIt was found that CAAs remodeled collagen alignment during crosstalk with breast cancer cells in vitro and in vivo, which further promoted breast cancer metastasis. Tumor-derived PAI-1 was required to activate the expression of the intracellular enzyme procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) in CAAs. Pharmacologic blockade of PAI-1 or PLOD2 disrupted the collagen reorganization in CAAs. Mechanistically, it was observed that PI3K/AKT pathway was activated in adipocytes upon co-culturing with breast cancer cells or treatment with recombinant PAI-1, which could promote the translocation of transcription factor FOXP1 into the nucleus and further enhanced the promoter activity of PLOD2 in CAAs. In addition, collagen reorganization at the tumor-adipose periphery, as well as the positive relevance between PAI-1 and PLOD2 in invasive breast carcinoma were confirmed in clinical specimens of breast cancer.ConclusionIn summary, our findings revealed a new stromal collagen network that favors tumor invasion and metastasis establish between breast cancer cells and surrounding adipocytes at the tumor invasive front, and identified PLOD2 as a therapeutic target for metastatic breast cancer treatment.

Dissecting the emerging role of cancer-associated adipocyte-derived cytokines in remodeling breast cancer progression

Background: Once breast carcinoma in situ breaks through the basement membrane, it preferentially invades the lipid-rich microenvironment around the mammary gland, which indicates a poor prognosis. However, the detailed mechanisms of this interaction remain elusive. Here, we describe bidirectional communication between breast cancer cells and cancer-associated adipocytes (CAAs). Different from the common hormone-driven mechanism of lipolysis of adipocytes, we found that CAAs perform a unique lipolysis program, and Zeb1 plays an important role in this lipolysis process. Methods: Fabp4-cre;Zeb1-/- as adipose tissue-specific Zeb1 gene knockout homozygous mice and Fabp4-cre;Zeb1 tg as adipose tissue-specific Zeb1 gene overexpression mice were constructed to study mammary gland development and perform orthotopic tumor transplantation experiments. Tetracycline-induced Zeb1-overexpressing stable cells were constructed from mouse preadipocytes 3T3-L1 to study phenotypes of lipolysis. Single-cell sequencing, targeted lipidomics, and RNA sequencing analysis were used to investigate the interaction between cancer cells and adipocytes. Western blotting and immunohistochemical staining assays were used to detect the expression Zeb1 at the cellular level, in mouse models and in patient tissue samples. Results: Our results indicated that breast cancer cell-derived adrenomedullin (ADM) significantly down-regulates Zeb1 expression in adipocytes, which drives downstream gene expression of Pnpla2/HSL/SCD1 and induces adipocyte lipolysis into CAA, a process that results in the release of excess palmitoleic acid. Consequently, we proved that palmitoleic acid could replace arachidonic acid (ARA) in the phospholipid sn-2 position of breast cell membrane, which enhances the fluidity and invasiveness of cancer cells. In addition, overexpression of Zeb1 in adipocytes reduces the ability of cancer cells to induce adipolysis and inhibits the malignant progression of cancer cells. Conclusion: These results indicate that Zeb1 plays a pivotal role as core transcriptional factor in CAA lipolysis. The interaction between breast cancer cells and adipocytes leads to the reprogramming of metabolism within their tumor microenvironment, which may become a new target for breast cancer treatment. Citation Format: Lixia Cao, Xiao Chen, Yi Shi, Shuang Yang. Cancer cell-derived adrenomedullin downregulates Zeb1 in adipocytes to induce adipolysis and promote breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4434.

IntroductionIn breast cancer, obesity is linked to invasive tumours that respond poorly to chemotherapy. We have explored relationships between breast cancer cells and adipocytes in the tumour microenvironment using a transwell co-culture system. Human breast adipocytes induced chemo-resistance in both hormone receptor positive (MCF-7) and triple negative (MDA-MB-231) breast cancer cells. In addition, cancer-associated adipocytes (CAA) induced a partial epithelial-mesenchymal transition, creating a population of breast cancer cells that were highly migratory. In this study, we used discovery mass spectrometry and antibody arrays to analyse the proteins secreted by CAA to identify proteins that may contribute to breast cancer progression.Material and methodsBreast adipose tissue samples were collected from patients undergoing breast surgery at Christchurch Hospital. Pre-adipocytes were isolated and differentiated into mature adipocytes, followed by co-culture with MCF-7 and MDA-MB-231 cancer cells for 3 days in a transwell co-culture system. Breast cancer cells were removed and adipocytes were washed and serum free media was added for 24 hours. This conditioned media was collected from pre-adipocytes, mature differentiated adipocytes and CAA co-cultured with either MCF-7 or MDA-MB-231 cells. Secretomes were compared using antibody arrays and iTRAQ labelling with LC-coupled LTQ-Orbitrap tandem mass spectrometry. Validation of iTRAQ results was performed using ELISA.Results and discussionsOf the 327 proteins identified using iTRAQ-MS, 11.3% were intracellular and thus excluded; the remainder were either extracellular or secreted proteins. Of the 183 secreted proteins, 45% of these were secreted at higher levels by adipocytes after co-culture with breast cancer cells compared with adipocytes alone. These included proteins associated with inflammation and matrix remodelling. Chitinase 3-like 1 (Chi3L1) levels were increased according to both iTRAQ-MS and antibody array analysis. Recombinant Chi3L1 treatment (4 ng/ml) increased viability, proliferation, migration and resistance to chemotherapy in both breast cancer cell lines.ConclusionOur study describes for the first time, the secretome profile from CAA and provides a comprehensive platform for further research investigating local interactions between breast cancer cells and CAA. Our data identified CAA derived Chi3L1 as a key player in the interactions between adipocytes and cancer cells within the tumour microenvironment.

Adipocyte is the most predominant cell type in the tumor microenvironment of breast cancer and plays a pivotal role in cancer progression, yet the underlying mechanisms and functional mediators remain elusive. We isolated primary preadipocytes from mammary fat pads of human breast cancer patients and generated mature adipocytes and cancer-associated adipocytes (CAAs) in vitro. The CAAs exhibited significantly different gene expression profiles as assessed by transcriptome sequencing. One of the highly expressed genes in CAAs is granulocyte colony-stimulating factor (G-CSF). Treatment with recombinant human G-CSF protein or stable expression of human G-CSF in triple-negative breast cancer (TNBC) cell lines enhanced epithelial–mesenchymal transition, migration, and invasion of cancer cells, by activating Stat3. Accordantly, targeting G-CSF/Stat3 signaling with G-CSF-neutralizing antibody, a chemical inhibitor, or siRNAs for Stat3 could all abrogate CAA- or G-CSF-induced migration and invasion of breast cancer cells. The pro-invasive genes MMP2 and MMP9 were identified as target genes of G-CSF in TNBC cells. Furthermore, in human breast cancer tissues, elevated G-CSF expression in adipocytes is well correlated with activated Stat3 signal in cancer cells. Together, our results suggest a novel strategy to intervene with invasive breast cancers by targeting CAA-derived G-CSF.

Runx1 activates the transformation of adipocytes into cancer-associated adipocytes by downregulating Plin1.

Simple SummaryBreast cancer is the most prevalent cancer in women worldwide, and it exhibits a growing incidence. An increasing number of studies showed the complex bidirectional regulation between breast cancer and adjacent cancer-associated adipocytes. The present review summarizes the mechanisms of cancer-associated adipocyte formation in the breast cancer tumor microenvironment and the effect of cancer-associated adipocytes on the tumorigenesis, progression, and metastasis of breast cancer. We focused on the therapeutic resistance of breast cancer caused by cancer-associated adipocytes and potential strategies targeting cancer-associated adipocytes in breast cancer treatment.Adipocytes are the main components in breast tissue, and cancer-associated adipocytes (CAAs) are one of the most important components in the tumor microenvironment of breast cancer (BC). Bidirectional regulation was found between CAAs and BC cells. BC facilitates the dedifferentiation of adjacent adipocytes to form CAAs with morphological and biological changes. CAAs increase the secretion of multiple cytokines and adipokines to promote the tumorigenesis, progression, and metastasis of BC by remodeling the extracellular matrix, changing aromatase expression, and metabolic reprogramming, and shaping the tumor immune microenvironment. CAAs are also associated with the therapeutic response of BC and provide potential targets in BC therapy. The present review provides a comprehensive description of the crosstalk between CAAs and BC and discusses the potential strategies to target CAAs to overcome BC treatment resistance.

Exploring new diagnostic biomarkers and molecular targets is of great importance in breast cancer treatment. The present study investigated the effects of acetyl-CoA carboxylase (ACC) expression interference on the malignant progression of breast cancer cells. ACC expression was knocked down using a lentiviral vector and this was verified by quantitative polymerase chain reaction and western blotting. MCF-7 and MDA-MB-231 breast cancer cells were randomly allocated into the following groups: Normal breast cancer cells (control), breast cancer cells transduced with a negative control lentiviral vector and breast cancer cells transduced with an ACC knockdown lentiviral vector. Screening for stable transgenic strains was successful. Cell viability, apoptosis and migration were determined using Cell Counting Kit-8, flow cytometry and scratch test, respectively. The protein expression levels of N-cadherin, Vimentin and Bax were detected by western blotting. In addition, a nude mouse model of subcutaneous metastatic tumor was established using MCF-7 breast cancer cells, and tumor volume was assessed. Furthermore, pathological condition and apoptosis were detected using hematoxylin and eosin, and TUNEL staining, respectively. The protein expression levels of N-cadherin, Vimentin and Bax were detected by western blotting. The in vitro experiments showed that knockdown of ACC expression significantly decreased the viability and migration, and increased the apoptosis of MCF-7 and MDA-MB-231 breast cancer cells. In vivo experiments revealed that ACC knockdown effectively reduced the tumor volume in nude mice, and promoted tumor cell apoptosis. Both in vitro and in vivo experiments showed that ACC knockdown can reduce the protein expression levels of N-cadherin and Vimentin, and increase Bax expression. These findings suggested that downregulation of ACC expression may significantly reduce the malignant progression of breast cancer, and could be considered a potential therapeutic target.

BackgroundAdipocytes are engaged in the development and progression of breast cancer (BC). Cancer-associated adipocytes (CAAs) are specific adipocytes located at the invasive front of BC that modulate tumor behaviors. This study aimed to investigate the effect of CAA-derived IL-6 in regulating BC progression.MethodsHuman BC specimens and adipocytes co-cultured with BC cells were used to explore the characteristics of CAAs. Adipocytes and 4T1 cells co-implanted in mouse model and tail vein metastasis model were used to explore the effect of CAAs on malignant progression and immunosuppressive tumor microenvironment (TME) of BC in vivo. The functional assays, flow cytometry, ELISA, miRNAs sequencing and dual-luciferase reporter assay were implemented to investigate the role of CAA-derived IL-6 in regulating programmed cell death protein ligand 1 (PD-L1) expression.ResultsCAAs were present at the invasive front of BC with a de-differentiated fibroblast phenotype. CAAs enhanced the malignant behaviors of 4T1 BC cells in vitro, and promoted 4T1 tumor growth and lung metastasis with decreased CD8+T cells and upregulated Tregs. The IHC results of both human BC specimens and xenografts showed that CAAs could upregulate PD-L1 expression in BC. Besides, CAAs could secrete abundant IL-6 and thus enhanced PD-L1 expression in 4T1 cells and tumors. More importantly, CAA-derived IL-6 could activate STAT3, while STAT3 blockade in CAA-cultured 4T1 cells upregulated miRNA-497a-5p expression and downregulated PD-L1 expression. Dual-luciferase reporter assay indicated that PD-L1 was a direct target of miRNA-497a-5p.ConclusionsOur study demonstrated that CAAs promoted the malignant behaviors of BC and enhanced immunosuppression in TME. Specifically, CAA-derived IL-6 promoted the PD-L1 expression of BC via STAT3/miR-497a-5p signaling, thereby contributing to BC progression.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00432-025-06324-5.

The role of adipocytes in cancer microenvironment has gained focus during the recent years. However, the characteristics of the cancer-associated adipocytes (CAA) in human breast cancer tissues and the underlying regulatory mechanism are not clearly understood. We reviewed pathology specimens of breast cancer patients to understand the morphologic characteristics of CAA, and profiled the mRNA and miRNA expression of CAA by using indirect co-culture system in vitro. The CAAs in human breast cancers showed heterogeneous topographic relationship with breast cancer cells within the breast microenvironment. The CAAs exhibited the characteristics of de-differentiation determined by their microscopic appearance and the expression levels of adipogenic markers. Additionally, the 3T3-L1 adipocytes indirectly co-cultured with breast cancer cells showed up-regulation of inflammation-related genes including Il6 and Ptx3. The up-regulation of IL6 in CAA was further observed in human breast cancer tissues. miRNA array of indirectly co-cultured 3T3-L1 cells showed increased expression of mmu-miR-5112 which may target Cpeb1. Cpeb1 is a negative regulator of Il6. The suppressive role of mmu-miR-5112 was confirmed by dual luciferase reporter assay, and mmu-miR-5112-treated adipocytes showed up-regulation of Il6. The transition of adipocytes into more inflammatory CAA resulted in proliferation-promoting effect in ER positive breast cancer cells such as MCF7 and ZR-75-1 but not in ER negative cells. In this study, we have determined the de-differentiated and inflammatory natures of CAA in breast cancer microenvironment. Additionally, we propose a miRNA-based regulatory mechanism underlying the process of acquiring inflammatory phenotypes in CAA.

Cancer-associated adipocytes (CAAs), which are adipocytes transformed by cancer cells, are of great importance in promoting the progression of breast cancer. However, the underlying mechanisms involved in the crosstalk between cancer cells and adipocytes are still unknown. Here we report that CAAs and breast cancer cells communicate with each other by secreting the cytokines leukemia inhibitory factor (LIF) and C-X-C subfamily chemokines (CXCLs), respectively. LIF is a pro-inflammatory cytokine secreted by CAAs, which promotes migration and invasion of breast cancer cells via the Stat3 signaling pathway. The activation of Stat3 induced the secretion of glutamic acid-leucine-arginine (ELR) motif CXCLs (CXCL1, CXCL2, CXCL3 and CXCL8) in tumor cells. Interestingly, CXCLs in turn activated the ERK1/2/NF-κB/Stat3 signaling cascade to promote the expression of LIF in CAAs. In clinical breast cancer pathology samples, the up-regulation of LIF in paracancerous adipose tissue was positively correlated with the activation of Stat3 in breast cancer. Furthermore, we verified that adipocytes enhanced lung metastasis of breast cancer cells, and the combination of EC330 (targeting LIF) and SB225002 (targeting C-X-C motility chemokine receptor 2 (CXCR2)) significantly reduced lung metastasis of breast cancer cells in vivo. Our findings reveal that the interaction of adipocytes with breast cancer cells depends on a positive feedback loop between the cytokines LIF and CXCLs, which promotes breast cancer invasion and metastasis.