The Lipid Phenotype of Breast Cancer Cells Characterized by Raman Microspectroscopy: Towards a Stratification of Malignancy

Although molecular classification brings interesting insights into breast cancer taxonomy, its implementation in daily clinical care is questionable because of its expense and the information supplied in a single sample allocation is not sufficiently reliable. New approaches, based on a panel of small molecules derived from the global or targeted analysis of metabolic profiles of cells, have found a correlation between activation of de novo lipogenesis and poorer prognosis and shorter disease-free survival for many tumors. We hypothesized that the lipid content of breast cancer cells might be a useful indirect measure of a variety of functions coupled to breast cancer progression. Raman microspectroscopy was used to characterize metabolism of breast cancer cells with different degrees of malignancy. Raman spectra from MDA-MB-435, MDA-MB-468, MDA-MB-231, SKBR3, MCF7 and MCF10A cells were acquired with an InVia Raman microscope (Renishaw) with a backscattered configuration. We used Principal Component Analysis and Partial Least Squares Discriminant Analyses to assess the different profiling of the lipid composition of breast cancer cells. Characteristic bands related to lipid content were found at 3014, 2935, 2890 and 2845 cm⁻¹, and related to lipid and protein content at 2940 cm⁻¹. A classificatory model was generated which segregated metastatic cells and non-metastatic cells without basal-like phenotype with a sensitivity of 90% and a specificity of 82.1%. Moreover, expression of SREBP-1c and ABCA1 genes validated the assignation of the lipid phenotype of breast cancer cells. Indeed, changes in fatty acid unsaturation were related with the epithelial-to-mesenchymal transition phenotype. Raman microspectroscopy is a promising technique for characterizing and classifying the malignant phenotype of breast cancer cells on the basis of their lipid profiling. The algorithm for the discrimination of metastatic ability is a first step towards stratifying breast cancer cells using this rapid and reagent-free tool.     

Estrogen promotes reversible epithelial-to-mesenchymal-like transition and collective motility in MCF-7 breast cancer cells

The role of estrogen in the motility and invasion of breast cancer cells is controversial. Although estrogen receptor (ER)-positive breast tumors are considered less aggressive and more differentiated they still undergo metastasis. In many types of epithelial cancers, the ability to undergo metastasis has been associated with a loss of epithelial features and acquisition of mesenchymal properties leading to migration of individual cells, a process known as epithelial-to-mesenchymal transition (EMT). In this report, we show that a subset of ER-positive breast cancer cells can acquire mesenchymal-like features and motility in a reversible manner. In MCF-7 breast cancer cells estrogen-promoted acquisition of mesenchymal-like features while antiestrogens, such as tamoxifen, prevented this transition. Moreover, pharmacological inhibition of Src family kinases decreased the ability of estrogen to promote epithelial-to-mesenchymal-like transition. In addition to mesenchymal-like motility, a subset of estrogen-treated cells also moved as cell clusters (collective motility). While membrane localization of E-cadherin/beta-catenin was decreased in fibroblast-like cells, enhanced levels of E-cadherin/beta-catenin were detected in motile cell clusters. Thus, during tumor progression, estrogen may foster motility and invasion of ER-positive breast cancer by promoting simultaneously reversible EMT-like changes and collective motility. These studies suggest that antiestrogen therapy and Src family kinase inhibitors may decrease development of metastases in ER-positive breast cancer by blocking estrogen-dependent migration of human breast cancer cells.     

Role of TRPV4 in matrix stiffness-induced expression of EMT-specific LncRNA

Molecular and Cellular Biochemistry - Human triple negative breast cancer cells, MDA-MB-231, show typical epithelial to mesenchymal transition associated with cancer progression. Mitochondria play...     

DGKZ promotes TGFβ signaling pathway and metastasis in triple-negative breast cancer by suppressing lipid raft-dependent endocytosis of TGFβR2

Diacylglycerol kinase ζ (DGKZ) is a diacylglycerol kinase that metabolizes diacylglycerol to yield phosphatidic acid, and its function in breast cancer progression remains unclear. In this study, via screening of a CRISPR-Cas9 knockout library containing lipid metabolic genes, DGKZ was identified as a potential prometastatic gene. We first confirmed that high DGKZ expression correlated with tumor progression and poor prognosis in patients. Next, knockout of DGKZ in triple-negative breast cancer cell lines were found to significantly inhibit metastatic behaviors in vitro and in vivo, whereas its overexpression increased the metastatic potential of cell lines. Mechanistic studies based on RNA sequencing and bioinformatic analysis indicated that DGKZ might regulate cell metastasis by promoting epithelial–mesenchymal transition via the transforming growth factor β (TGFβ) signaling pathway. Furthermore, we found that overexpression of DGKZ activated the TGFβ/TGFβR2/Smad3 signaling pathway by inhibiting the degradation of TGFβR2 through suppression of caveolin/lipid raft-dependent endocytosis. Moreover, the caveolin/lipid raft-dependent endocytosis of TGFβR2 was regulated by the metabolite phosphatidic acid, which might alter TGFβR2 partitioning in lipid rafts and nonlipid rafts by affecting the fluidity of the plasma membrane. These findings suggested that DGKZ is a novel promoter of metastasis and that it could be a potential prognostic indicator in patients with triple-negative breast cancer.Subject terms: Breast cancer, Cell migration     

Tumor grafts derived from women with breast cancer authentically reflect tumor pathology, growth, metastasis and disease outcomes

Development and preclinical testing of new cancer therapies is limited by the scarcity of in vivo models that authentically reproduce tumor growth and metastatic progression. We report new models for breast tumor growth and metastasis in the form of transplantable tumors derived directly from individuals undergoing treatment for breast cancer. These tumor grafts illustrate the diversity of human breast cancer and maintain essential features of the original tumors, including metastasis to specific sites. Co-engraftment of primary human mesenchymal stem cells maintains phenotypic stability of the grafts and increases tumor growth by promoting angiogenesis. We also report that tumor engraftment is a prognostic indicator of disease outcome for women with newly diagnosed breast cancer; orthotopic breast tumor grafting is a step toward individualized models for tumor growth, metastasis and prognosis. This bank of tumor grafts also serves as a publicly available resource for new models in which to study the biology of breast cancer.     

MIR-99a and MIR-99b modulate TGF-β induced epithelial to mesenchymal plasticity in normal murine mammary gland cells

Epithelial to mesenchymal transition (EMT) is a key process during embryonic development and disease development and progression. During EMT, epithelial cells lose epithelial features and express mesenchymal cell markers, which correlate with increased cell migration and invasion. Transforming growth factor-β (TGF-β) is a multifunctional cytokine that induces EMT in multiple cell types. The TGF-β pathway is regulated by microRNAs (miRNAs), which are small non-coding RNAs regulating the translation of specific messenger RNAs.Herein, we identified mir-99a and mir-99b as two novel TGF-β target miRNA genes, the expression of which increased during TGF-β induced EMT of NMUMG cells. Mir-99a and mir-99b inhibition decreased TGF-β activity by inhibiting SMAD3 phosphorylation, resulting in decreased migration and increased proliferation in response to TGF-β. However, mir-99a and mir-99b inhibition was insufficient to block TGF-β induced EMT of NMUMG cells.Mir-99a and mir-99b over-expression in epithelial NMUMG cells resulted in increased proliferation, migration and fibronectin expression, while E-cadherin and ZO-1 expression were negatively regulated.In conclusion, we identified mir-99a and mir-99b as two novel modulators of TGF-β pathway that alter SMAD3 phosphorylation, in turn altering cell migration and adhesion of mesenchymal NMUMG cells. The effect of mir-99a and mir-99b over-expression on NMUMUG proliferation is dependent upon the epithelial or mesenchymal status of the cells. Our study suggests that mir-99a and mir-99b may function as modulators within a complex network of factors regulating TGF-β induced breast epithelial to mesenchymal transition, as well as proliferation and migration of breast cancer cells, providing a possible target for future translationally oriented studies in this area.     

HAO2 inhibits malignancy of clear cell renal cell carcinoma by promoting lipid catabolic process

Hydroxy acid oxidase 2 (HAO2) has been reported to inhibit tumor progression through metabolic pathway. The current study was designed to evaluate the prognostic significance and probable mechanism of HAO2 in patients with clear cell renal cell carcinoma (ccRCC). The study screened The Cancer Genome Atlas Kidney Clear Cell Carcinoma (TCGA-KIRC) database for patients with ccRCC having complete clinical information and HAO2 expression. Low HAO2 was associated with shorter overall survival (OS) and shorter disease-free survival (DFS). Gene set enrichment analysis (GSEA) showed HAO2 was associated with neutral lipid catabolic process, metabolic process, lipid oxidation, epithelial–mesenchymal transition (EMT), and Kirsten rat sarcoma viral oncogene signaling (KRAS). Western blot analysis and immunohistochemistry analysis checked HAO2 expression in ccRCC cancer tissues, normal tissues, and renal cancer cell lines. HAO2 was downregulated in ccRCC cancer tissues and ccRCC cell lines when compared with their control group. Overexpression of HAO2 by plasmid promoted lipid catabolic process, eliminated lipid accumulation, inhibited KRAS expression, controlled the proliferation, migration, and invasion activity of ccRCC tumor cells. Our results indicated that HAO2 inhibits malignancy ccRCC by promoting lipid catabolic process, HAO2 could be an effective molecular marker and treatment for ccRCC.     

Breast Cancer‐Derived Exosomes Reflect the Cell‐of‐Origin Phenotype

A manner in which cells can communicate with each other is via secreted nanoparticles termed exosomes. These vesicles contain lipids, nucleic acids, and proteins, and are said to reflect the cell‐of‐origin. However, for the exosomal protein content, there is limited evidence in the literature to verify this statement. Here, proteomic assessment combined with pathway‐enrichment analysis is used to demonstrate that the protein cargo of exosomes reflects the epithelial/mesenchymal phenotype of secreting breast cancer cells. Given that epithelial‐mesenchymal plasticity is known to implicate various stages of cancer progression, the results suggest that breast cancer subtypes with distinct epithelial and mesenchymal phenotypes may be distinguished by directly assessing the protein content of exosomes. Additionally, the work is a substantial step toward verifying the statement that cell‐derived exosomes reflect the phenotype of the cells‐of‐origin.     

MeCP2 inhibits proliferation and migration of breast cancer via suppression of epithelial‐mesenchymal transition

Methyl‐CpG‐binding protein 2 (MeCP2) is an important epigenetic regulator for normal neuronal maturation and brain glial cell function. Additionally, MeCP2 is also involved in a variety of cancers, such as breast, prostate, lung, liver and colorectal. However, whether MeCP2 contributes to the progression of breast cancer remains unknown. In the present study, we investigated the role of MeCP2 in cell proliferation, migration and invasion in vitro. We found that knockdown of MeCP2 inhibited expression of epithelial‐mesenchymal transition (EMT)‐related markers in breast cancer cell lines. In conclusion, our study suggests that MeCP2 inhibits proliferation and invasion through suppression of the EMT pathway in breast cancer.     

Proteomic identification of glycosylphosphatidylinositol anchor-dependent membrane proteins elevated in breast carcinoma

The glycosylphosphatidylinositol (GPI) anchor is a lipid and glycan modification added to the C terminus of certain proteins in the endoplasmic reticulum by the activity of a multiple subunit enzyme complex known as the GPI transamidase (GPIT). Several subunits of GPIT have increased expression levels in breast carcinoma. In an effort to identify GPI-anchored proteins and understand the possible role of these proteins in breast cancer progression, we employed a combination of strategies. First, alpha toxin from Clostridium septicum was used to capture GPI-anchored proteins from human breast cancer tissues, cells, and serum for proteomic analysis. We also expressed short interfering RNAs targeting the expression of the GPAA1 and PIGT subunits of GPIT in breast cancer cell lines to identify proteins in which membrane localization is dependent on GPI anchor addition. Comparative membrane proteomics using nano-ESI-RPLC-MS/MS led to the discovery of several new potential diagnostic and therapeutic targets for breast cancer. Furthermore, we provide evidence that increased levels of GPI anchor addition in malignant breast epithelial cells promotes the dedifferentiation of malignant breast epithelial cells in part by increasing the levels of cell surface markers associated with mesenchymal stem cells.     

Crosstalk between ferroptosis and the epithelial-mesenchymal transition: Implications for inflammation and cancer therapy

Both genomic instability and the presence of chronic inflammation are involved in carcinogenesis and tumor progression. These alterations predispose the cancer cells to undergo metabolic reprogramming as well as the epithelial-mesenchymal transition (EMT). These pathways allow cancer cells to avoid apoptosis and stimulate tumor progression. EMT is an important early event in tumor cell invasion, which can be regulated through inflammatory signaling pathways. Cancer cells undergoing EMT are vulnerable to cell death by the process of ferroptosis. Ferroptosis is a form of regulated cell death involving iron-dependent lipid peroxidation, designed to maintain cellular homeostasis. Several reports have linked ferroptosis, inflammation, and cancer. Ferroptosis inhibitors and EMT inducers have been used to understand the anti-inflammatory and anticancer effects in experimental models. A better understanding of the crosstalk between ferroptosis and EMT, and the involvment of inflammatory mediators may accelerate the discovery of therapeutic strategies to eradicate cancer cells and overcome drug-resistance.     

糖代谢重编程在肿瘤上皮-间质转变过程中的生物学意义

上皮-间质转变(epithelial to mesenchymal transition,EMT)即成熟组织中的上皮细胞转变成具有迁移能力的间质细胞,该过程在恶性肿瘤的侵袭和转移过程中发挥重要作用。随着肿瘤的发生发展,细胞在恶性转变的同时往往会发生代谢方式的重编程,从依赖线粒体氧化磷酸化产生ATP的代谢方式转变为通过糖酵解途径吸收和利用葡萄糖。本文主要对肿瘤糖代谢重编程与细胞EMT转变关系的研究进展进行了综述。     

Epithelial cell adhesion molecule promotes breast cancer resistance protein-mediated multidrug resistance in breast cancer by inducing partial epithelial–mesenchymal transition

Overexpression of breast cancer resistance protein (BCRP) plays a crucial role in the acquired multidrug resistance (MDR) in breast cancer. The elucidation of molecular events that confer BCRP-mediated MDR is of major therapeutic importance in breast cancer. Epithelial cell adhesion molecule (EpCAM) has been implicated in tumor progression and drug resistance in various types of cancers, including breast cancer. However, the role of EpCAM in BCRP-mediated MDR in breast cancer remains unknown. In the present study, we revealed that EpCAM expression was upregulated in BCRP-overexpressing breast cancer MCF-7/MX cells, and EpCAM knockdown using siRNA reduced BCRP expression and increased the sensitivity of MCF-7/MX cells to mitoxantrone (MX). The epithelial–mesenchymal transition (EMT) promoted BCRP-mediated MDR in breast cancer cells, and EpCAM knockdown partially suppressed EMT progression in MCF-7/MX cells. In addition, Wnt/β-catenin signaling was activated in MCF-7/MX cells, and the inhibition of this signaling attenuated EpCAM and BCRP expression and partially reversed EMT. Together, this study illustrates that EpCAM upregulation by Wnt/β-catenin signaling induces partial EMT to promote BCRP-mediated MDR resistance in breast cancer cells. EpCAM may be a potential therapeutic target for overcoming BCRP-mediated resistance in human breast cancer.