Cancer-associated Fibroblasts Formation Research Articles

Mechanotransduction-The relationship between gravity, cells and tensile loading in extracellular matrix

Gravity plays a central role in vertebrate development and evolution. Mechanotransduction involves the tensile tethering of veins and arteries, connections between the epidermis and dermis in skin, tensile stress concentrations that occur at tissue interfaces, cell-cell interactions, cell-collagen fiber stress transfer in extracellular matrix and fluid shear flow. While attention in the past has been directed at understanding the myriad of biochemical players associated with mechanotransduction pathways, less attention has been focused on determining the tensile mechanical behavior of tissues in vivo. Fibroblasts sit on the surface of collagen fibers in living skin and exert a retractile force on the fibers. This retractile force pulls against the tension in collagen fibers in skin. After fibroblast-collagen fiber interactions are altered either by changes in fibroblast adhesion or after formation of cancer associated fibroblasts, and changes in cell junctions, alterations in the retractive force leads to changes in mechanotransduction. The purpose of this paper is to present a model of tensile forces that occur at the fibroblast-collagen fiber interface and how these forces are important in extracellular matrix physiology in health and disease.

Smad3 Promotes Cancer-Associated Fibroblasts Generation via Macrophage-Myofibroblast Transition.

Cancer‐associated fibroblasts (CAFs) are important in tumor microenvironment (TME) driven cancer progression. However, CAFs are heterogeneous and still largely underdefined, better understanding their origins will identify new therapeutic strategies for cancer. Here, the authors discovered a new role of macrophage‐myofibroblast transition (MMT) in cancer for de novo generating protumoral CAFs by resolving the transcriptome dynamics of tumor‐associated macrophages (TAM) with single‐cell resolution. MMT cells (MMTs) are observed in non‐small‐cell lung carcinoma (NSCLC) associated with CAF abundance and patient mortality. By fate‐mapping study, RNA velocity, and pseudotime analysis, existence of novel macrophage‐lineage‐derived CAF subset in the TME of Lewis lung carcinoma (LLC) model is confirmed, which is directly transited via MMT from M2‐TAM in vivo and bone‐marrow‐derived macrophages (BMDM) in vitro. Adoptive transfer of BMDM‐derived MMTs markedly promote CAF formation in LLC‐bearing mice. Mechanistically, a Smad3‐centric regulatory network is upregulated in the MMTs of NSCLC, where chromatin immunoprecipitation sequencing(ChIP‐seq) detects a significant enrichment of Smad3 binding on fibroblast differentiation genes in the macrophage‐lineage cells in LLC‐tumor. More importantly, macrophage‐specific deletion and pharmaceutical inhibition of Smad3 effectively block MMT, therefore, suppressing the CAF formation and cancer progression in vivo. Thus, MMT may represent a novel therapeutic target of CAF for cancer immunotherapy.

The Role of Non-coding Genome in Cancer-associated Fibroblasts; Stateof- the-Art and Perspectives in Cancer Targeted Therapy.

Cancer-associated fibroblasts (CAFs) are senescent fibroblasts in tumor nest, which trigger a signaling center to remodel a desmoplastic tumor niche. CAF's functions in cancer are closely similar to myofibroblasts during the wound healing process. They can produce cytokines, enzymes, and protein- or RNA-containing exosomes to alter the function of surrounding cells. Non-- coding RNAs, including microRNAs and long non-coding RNAs, modulate pathologic mechanisms in cancer. Dysregulation of these RNAs influences the formation and function of CAFs. Furthermore, it has been demonstrated that CAFs, by releasing non-coding RNAs-containing exosomes, affect the tumor cells' behavior. CAFs also secrete mediators such as chemokines to alter the expression of non-coding RNAs in the tumor microenvironment. This study aimed to discuss the role of non-coding RNAs in CAF development in cancer. Additionally, we have shed light on the therapeutic approaches to develop the strategies based on the alteration of non-coding RNAs in cancer.

Regulation of Extracellular Matrix Production in Activated Fibroblasts: Roles of Amino Acid Metabolism in Collagen Synthesis.

Cancer associated fibroblasts (CAFs) are a major component of the tumour microenvironment in most tumours, and are key mediators of the response to tissue damage caused by tumour growth and invasion, contributing to the observation that tumours behave as ‘wounds that do not heal’. CAFs have been shown to play a supporting role in all stages of tumour progression, and this is dependent on the highly secretory phenotype CAFs develop upon activation, of which extracellular matrix (ECM) production is a key element. A collagen rich, stromal ECM has been shown to influence tumour growth and metastasis, exclude immune cells and impede drug delivery, and is associated with poor prognosis in many cancers. CAFs also extensively remodel their metabolism to support cancer cells, however, it is becoming clear that metabolic rewiring also supports intrinsic functions of activated fibroblasts, such as increased ECM production. In this review, we summarise how fibroblasts metabolically regulate ECM production, focussing on collagen production, at the transcriptional, translational and post-translational level, and discuss how this can provide possible strategies for effectively targeting CAF activation and formation of a tumour-promoting stroma.

LAMC1 upregulation via TGFβ induces inflammatory cancer-associated fibroblasts in esophageal squamous cell carcinoma via NF-κB-CXCL1-STAT3.

Cancer‐associated fibroblasts (CAF) are a heterogeneous cell population within the tumor microenvironment,and play an important role in tumor development. By regulating the heterogeneity of CAF, transforming growth factor β (TGFβ) influences tumor development. Here, we explored oncogenes regulated by TGFβ1 that are also involved in signaling pathways and interactions within the tumor microenvironment. We analyzed sequencing data of The Cancer Genome Atlas (TCGA) and our own previously established RNA microarray data (GSE53625), as well as esophageal squamous cell carcinoma (ESCC) cell lines with or without TGFβ1 stimulation. We then focused on laminin subunit gamma 1 (LAMC1), which was overexpressed in ESCC cells, affecting patient prognosis, which could be upregulated by TGFβ1 through the synergistic activation of SMAD family member 4 (SMAD4) and SP1. LAMC1 directly promoted the proliferation and migration of tumor cells, mainly via Akt–NFκB–MMP9/14 signaling. Additionally, LAMC1 promoted CXCL1 secretion, which stimulated the formation of inflammatory CAF (iCAF) through CXCR2–pSTAT3. Inflammatory CAF promoted tumor progression. In summary, we identified the dual mechanism by which the upregulation of LAMC1 by TGFβ in tumor cells not only promotes ESCC proliferation and migration, but also indirectly induces carcinogenesis by stimulating CXCL1 secretion to promote the formation of iCAF. This finding suggests that LAMC1 could be a potential therapeutic target and prognostic marker for ESCC.

PDGF-BB regulates the transformation of fibroblasts into cancer-associated fibroblasts via the lncRNA LURAP1L-AS1/LURAP1L/IKK/IκB/NF-κB signaling pathway.

The most abundant cells in the tumor microenvironment are cancer-associated fibroblasts (CAFs). They play an important role in oral squamous cell carcinoma (OSCC) angiogenesis, invasion and metastasis. Platelet-derived growth factor (PDGF)-BB has an obvious regulating effect on the formation of CAFs through binding to PDGF receptor (PDGFR)-β, but the role of long non-coding (lnc)RNA in PDGF-BB-induced transformation of fibroblasts into CAFs remains poorly understood. Using an lncRNA ChIP, 370 lncRNA transcripts were identified to be significantly and differentially expressed between fibroblasts and PDGF-BB-induced fibroblasts, including 240 upregulated lncRNAs and 130 downregulated lncRNAs, indicating that lncRNAs are involved in the regulation of the transformation of CAFs. Previous studies have shown that the nuclear factor (NF)-κB signaling pathway plays an important role in the activation of CAFs. Dual-luciferase reporter assay and co-immunoprecipitation were conducted to confirm that the leucine-rich adaptor protein 1-like (LURAP1L), which is the target of lncRNA LURAP1L antisense RNA 1 (LURAP1L-AS1) had a positive regulatory effect on I-κB kinase (IKK)/NF-κB signaling. Therefore, LURAP1L-AS1 was selected and PDGF-BB was demonstrated to upregulate the expression of LURAP1L-AS1 and LURAP1L, which was reversed by a PDGFR-β inhibitor. Subsequently, knocking down LURAP1L-AS1 suppressed the expression of PDGF-BB-induced fibroblast activation marker protein α-smooth muscle actin, fibroblast activation protein-α, PDGFR-β and phosphorylated (p)-PDGFR-β. IKKα, p-IĸB and p-NF-κB were downregulated by the knockdown of LURAP1L-AS1 and upregulated by overexpression of LURAP1L-AS1. The present study indicates that LURAP1L-AS1/LURAP1L/IKK/IĸB/NF-κB plays an important regulatory role in PDGF-BB-induced fibroblast activation and may become a potential target for the treatment of OSCC.

PLAU directs conversion of fibroblasts to inflammatory cancer-associated fibroblasts, promoting esophageal squamous cell carcinoma progression via uPAR/Akt/NF-\u03baB/IL8 pathway

Cancer-associated fibroblasts (CAFs) plays an important role in the tumor microenvironment. The heterogeneity of CAFs affects the effect of CAFs on promoting or inhibiting tumors, which can be regulated by other cells in the tumor microenvironment through paracrine methods. The urokinase-type plasminogen activator (PLAU) system mediates cell proliferation, migration, adhesion, and other functions through the proteolytic system, intracellular signal transduction, and chemokine activation. PLAU promotes tumor progression in many tumors. We explored the function of PLAU in ESCC and the influence of PLAU secreted by tumor cells on the heterogeneity of CAFs. We found that PLAU is highly expressed in ESCC, which is related to poor prognosis and can be used as a prognostic marker for ESCC. Through loss-of function and gain-of function experiments, we found that PLAU promoted ESCC proliferation and clone formation via MAPK pathway, and promotes migration by upregulating Slug and MMP9, which can be reversed by the MEK 1/2 inhibitor U0126. At the same time, through sequencing, cytokine detection, and RT-qPCR verification, we found that tumor cells secreted PLAU promoted the conversion of fibroblasts to inflammatory CAFs, which upregulated expression and secretion of IL8 via the uPAR/Akt/NF-κB pathway. The IL8 secreted by CAFs in turn promotes the high expression of PLAU in tumor cells and further promoted the progression of ESCC. In summary, PLAU was not only a prognostic marker of ESCC, which promoted tumor cell proliferation and migration, but also promoted the formation of inflammatory CAFs by the PLAU secreted by tumor cells.

Dysregulation of Transcription Factor Activity During Formation of Cancer-Associated Fibroblasts.

The reciprocal interactions between cancer cells and the quiescent fibroblasts leading to the activation of cancer-associated fibroblasts (CAFs) serve an important role in cancer progression. Here, we investigated the activation of transcription factors (TFs) in prostate fibroblasts (WPMY cell line) co-cultured with normal prostate or tumorous cells (RWPE1 and RWPE2 cell lines, respectively). After indirect co-cultures, we performed mRNA-seq and predicted TF activity using mRNA expression profiles with the Systems EPigenomics Inference of Regulatory Activity (SEPIRA) package and the GTEx and mRNA-seq data of 483 cultured fibroblasts. The initial differential expression analysis between time points and experimental conditions showed that co-culture with normal epithelial cells mainly promotes an inflammatory response in fibroblasts, whereas with the cancerous epithelial, it stimulates transformation by changing the expression of the genes associated with microfilaments. TF activity analysis revealed only one positively regulated TF in the RWPE1 co-culture alone, while we observed dysregulation of 45 TFs (7 decreased activity and 38 increased activity) uniquely in co-culture with RWPE2. Pathway analysis showed that these 45 dysregulated TFs in fibroblasts co-cultured with RWPE2 cells may be associated with the RUNX1 and PTEN pathways. Moreover, we showed that observed dysregulation could be associated with FER1L4 expression. We conclude that phenotypic changes in fibroblast responses to co-culturing with cancer epithelium result from orchestrated dysregulation of signaling pathways that favor their transformation and motility rather than proinflammatory status. This dysregulation can be observed both at the TF and transcriptome levels.

Abstract LB-211: Synergistic anti-metastatic effect of pembrolizumab in combination with a novel mTORC1 inhibitor/AMPK agonist in a CAM metastatic triple negative breast cancer model

Abstract Triple negative breast cancer (TNBC) is highly aggressive and has higher rates of recurrence, resistance, and death than other breast cancers. Ten year survival rate is < 50%. Changes in the tumor stroma create an environment that provides support for tumor growth, progression, invasion and metastasis. The most prominent cell type in the tumor stroma is an activated form of fibroblasts, known as cancer associated fibroblasts (CAFs). In breast cancer, as much as 80% of stromal fibroblasts acquire the CAF phenotype secreting growth factors, hormones, and cytokines that act upon both the tumor and the stroma to promote the metastatic phenotype. NovoMedix has developed a novel mTORC1 inhibitor/AMPK agonist (NMIC9) that inhibits tumor cell proliferation, migration (98% decrease, p=0.0002), and invasion; and it prevents activation of the tumor stroma and formation of CAFs. NMIC9, a small molecule, has demonstrated oral efficacy as a single agent in mouse models of TNBC (93% reduction in tumor growth, p<0.005, with no significant tumor regrowth after treatment cessation) and safety in non-GLP toxicology studies in rats (NOAEL > 1000 mg/kg p.o.). Further, NMIC9 has no hERG effects, no CYP interactions, no mutagenicity in the AMES assay, and no kinase inhibition. Many TNBC therapies cause cardiomyopathy leading to heart failure. In primary mouse adult ventricular cardiomyocytes, addition of NMIC9 significantly reduces DOX-induced cardiomyocyte apoptosis and death (p<0.001) while increasing efficacy in TNBC cells. Further, daily treatment with NMIC9 at therapeutic levels for 8 weeks did not cause any changes in cardiac structure or function, indicating that there are no cardiac liabilities. Inhibition of metastases is particularly relevant for TNBC because it is the main cause of mortality. While immune checkpoint inhibitors have shown remarkable response rates in several types of cancer, the response rate in TNBC is only 5 – 30%. A drug that could increase efficacy of immune checkpoint inhibitors and decrease metastasis in TNBC could dramatically improve outcomes for these patients. Recent data in a chicken chorioallantoic membrane model with a highly aggressive TNBC cell line (MDA-MB-231) show that NMIC9 decreases tumor growth and metastasis alone and in combination with pembrolizumab. Tumor regression was 20% for pembrolizumab (ns), 30% for NMIC9 (p<0.05), and 64% for pembrolizumab + NMIC9 (p<0.005). Decrease in metastasis invasion was -0.08% (ns) for pembrolizumab, 49% (p<0.05) for NMIC9, and 64% (p<0.005) for pembrolizumab + NMIC9. Therefore, NMIC9 is a promising first in class drug, which when used in combination with pembrolizumab can significantly reduce tumor growth and metastasis and improve survival rates in TNBC. Citation Format: Leah Fung, Laura G. Corral, Robert W. Sullivan, Kyle W. Chan, Cathy A. Swindlehurst. Synergistic anti-metastatic effect of pembrolizumab in combination with a novel mTORC1 inhibitor/AMPK agonist in a CAM metastatic triple negative breast cancer model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-211.

TNF-α enhances TGF-β-induced endothelial-to-mesenchymal transition via TGF-β signal augmentation.

The tumor microenvironment (TME) consists of various components including cancer cells, tumor vessels, cancer‐associated fibroblasts (CAFs), and inflammatory cells. These components interact with each other via various cytokines, which often induce tumor progression. Thus, a greater understanding of TME networks is crucial for the development of novel cancer therapies. Many cancer types express high levels of TGF‐β, which induces endothelial‐to‐mesenchymal transition (EndMT), leading to formation of CAFs. Although we previously reported that CAFs derived from EndMT promoted tumor formation, the molecular mechanisms underlying these interactions remain to be elucidated. Furthermore, tumor‐infiltrating inflammatory cells secrete various cytokines, including TNF‐α. However, the role of TNF‐α in TGF‐β‐induced EndMT has not been fully elucidated. Therefore, this study examined the effect of TNF‐α on TGF‐β‐induced EndMT in human endothelial cells (ECs). Various types of human ECs underwent EndMT in response to TGF‐β and TNF‐α, which was accompanied by increased and decreased expression of mesenchymal cell and EC markers, respectively. In addition, treatment of ECs with TGF‐β and TNF‐α exhibited sustained activation of Smad2/3 signals, which was presumably induced by elevated expression of TGF‐β type I receptor, TGF‐β2, activin A, and integrin αv, suggesting that TNF‐α enhanced TGF‐β‐induced EndMT by augmenting TGF‐β family signals. Furthermore, oral squamous cell carcinoma‐derived cells underwent epithelial‐to‐mesenchymal transition (EMT) in response to humoral factors produced by TGF‐β and TNF‐α‐cultured ECs. This EndMT‐driven EMT was blocked by inhibiting the action of TGF‐βs. Collectively, our findings suggest that TNF‐α enhances TGF‐β‐dependent EndMT, which contributes to tumor progression.

The interplay between cancer associated fibroblasts and immune cells in the context of radiation therapy.

Fibroblasts are a key component of the tumor microenvironment (TME) that can serve as a scaffold for tumor cell migration and augment the tumor's ability to withstand harsh conditions. When activated by external or endogenous stimuli, normal fibroblasts become cancer associated fibroblasts (CAFs), a heterogeneous group of stromal cells in the tumor that are phenotypically and epigenetically different from normal fibroblasts. Dynamic crosstalk between cancer cells, immune cells, and CAFs through chemokines and surface signaling makes the TME conducive to tumor growth. When activated, CAFs promote tumorigenesis and metastasis through several phenomena including regulation of tumor immunity, metabolic reprogramming of the TME, extracellular matrixremodeling and contraction, and induction of therapeutic resistance. Ionizing radiation (radiation theraphy [RT]) is a potent immunological stimulant that has been shown to increase cytotoxic Teff infiltration and IFN-I stimulated genes. RT, however, is unable to overcome the infiltration and activation of immunosuppressive cells which can contribute to tumor progression. Another paradox of RT is that, while very effective at killing cancer cells, it can contribute to the formation of CAFs. This review examines how the interplay between CAFs and immune cells during RT contributes to organ fibrosis, immunosuppression, and tumor growth. We focus on targeting mechanistic pathways of CAF formation as a potentially effective strategy not only for preventing organ fibrosis, but also in hampering tumor progression in response to RT.

Lactate-mediated epigenetic reprogramming regulates formation of human pancreatic cancer-associated fibroblasts.

Even though pancreatic ductal adenocarcinoma (PDAC) is associated with fibrotic stroma, the molecular pathways regulating the formation of cancer associated fibroblasts (CAFs) are not well elucidated. An epigenomic analysis of patient-derived and de-novo generated CAFs demonstrated widespread loss of cytosine methylation that was associated with overexpression of various inflammatory transcripts including CXCR4. Co-culture of neoplastic cells with CAFs led to increased invasiveness that was abrogated by inhibition of CXCR4. Metabolite tracing revealed that lactate produced by neoplastic cells leads to increased production of alpha-ketoglutarate (aKG) within mesenchymal stem cells (MSCs). In turn, aKG mediated activation of the demethylase TET enzyme led to decreased cytosine methylation and increased hydroxymethylation during de novo differentiation of MSCs to CAF. Co-injection of neoplastic cells with TET-deficient MSCs inhibited tumor growth in vivo. Thus, in PDAC, a tumor-mediated lactate flux is associated with widespread epigenomic reprogramming that is seen during CAF formation.

Cancer-derived exosomes trigger endothelial to mesenchymal transition followed by the induction of cancer-associated fibroblasts

Cancer-associated fibroblasts (CAFs) play a pivotal role in tumor growth, but very little has been known about its characteristics and origin. Recently, cancer-derived exosome has been suggested to transdifferentiate CAFs, by a new mechanism of endothelial to mesenchymal transition (EndMT), initiating angiogenic processes and triggering metastatic evolution. However, an enabling tool in vitro is yet to be developed to investigate complicated procedures of the EndMT and the transdifferentiation under reconstituted tumor microenvironment. Here we proposed an in vitro microfluidic model which enables to monitor a synergetic effect of complex tumor microenvironment in situ, including extracellular matrix (ECM), interstitial flow and environmental exosomes. The number of CAFs differentiated from human umbilical vein endothelial cells (HUVECs) increased with melanoma-derived exosomes, presenting apparent morphological and molecular changes with pronounced motility. Mesenchymal stem cell (MSC)-derived exosomes were found to suppress EndMT, induce angiogenesis and maintain vascular homeostasis, while cancer-derived exosomes promoted EndMT. Capabilities of the new microfluidic model exist in precise regulation of the complex tumor microenvironment and therefore successful reconstitution of 3D microvasculature niches, enabling in situ investigation of EndMT procedure between various cell types. Statement of SignificanceThis study presents an in vitro 3D EndMT model to understand the progress of the CAF generation by recapitulating the 3D tumor microenvironment in a microfluidic device. Both cancer-derived exosomes and interstitial fluid flow synergetically played a pivotal role in the EndMT and consequent formation of CAFs through a collagen-based ECM. Our approach also enabled the demonstration of a homeostatic capability of MSC-derived exosomes, ultimately leading to the recovery of CAFs back to endothelial cells. The in vitro 3D EndMT model can serve as a powerful tool to validate exosomal components that could be further developed to anti-cancer drugs.

Persistent DNA strand breaks induce a CAF-like phenotype in normal fibroblasts.

Cancer-associated fibroblasts (CAFs) are an emerging target for cancer therapy as they promote tumour growth and metastatic potential. However, CAF targeting is complicated by the lack of knowledge-based strategies aiming to selectively eliminate these cells. There is a growing body of evidence suggesting that a pro-inflammatory microenvironment (e.g. ROS and cytokines) promotes CAF formation during tumorigenesis, although the exact mechanisms involved remain unclear. In this study, we reveal that a prolonged pro-inflammatory stimulation causes a de facto deficiency in base excision repair, generating unrepaired DNA strand breaks and thereby triggering an ATF4-dependent reprogramming of normal fibroblasts into CAF-like cells. Based on the phenotype of in vitro-generated CAFs, we demonstrate that midostaurin, a clinically relevant compound, selectively eliminates CAF-like cells deficient in base excision repair and prevents their stimulatory role in cancer cell growth and migration.

Effect of cancer-associated fibroblasts on radiosensitivity of cancer cells.

Solid tumors are composed of tumor epithelial cells and the stroma, which are seemingly separate but actually related through cell-cell and cell-matrix interactions. These interactions can promote tumor evolution. Cancer-associated fibroblasts (CAFs) are the most abundant non-neoplastic cells in the stroma and also among the most important cell types interacting with cancer cells. Particularly, cancer cells promote the formation and maintenance of CAFs by secreting various cytokines. The activated CAFs then synthesize a series of growth factors to promote tumor cell growth, invasion and metastasis. More importantly, the presence of CAFs also interferes with therapeutic efficacy, bringing severe challenges to radiotherapy. This review summarizes the effect of CAFs on the radiosensitivity of tumor cells and underscores the need for further studies on CAFs in order to improve the efficacy of antitumor therapy.

Melanoma miRNA trafficking controls tumour primary niche formation.

Melanoma originates in the epidermis and becomes metastatic after invasion into the dermis. Prior interactions between melanoma cells and dermis are poorly studied. Here, we show that melanoma cells directly affect the formation of the dermal tumour niche by microRNA trafficking before invasion. Melanocytes, cells of melanoma origin, are specialized in releasing pigment vesicles, termed melanosomes. In melanoma in situ, we found melanosome markers in distal fibroblasts before melanoma invasion. The melanosomes carry microRNAs into primary fibroblasts triggering changes, including increased proliferation, migration and pro-inflammatory gene expression, all known features of cancer-associated fibroblasts (CAFs). Specifically, melanosomal microRNA-211 directly targets IGF2R and leads to MAPK signalling activation, which reciprocally encourages melanoma growth. Melanosome release inhibitor prevented CAF formation. Since the first interaction of melanoma cells with blood vessels occurs in the dermis, our data suggest an opportunity to block melanoma invasion by preventing the formation of the dermal tumour niche.

Metabolic reprogramming of cancer-associated fibroblasts by IDH3α downregulation.

Cancer-associated fibroblasts (CAFs) provide critical metabolites for tumor growth and undergo metabolic reprogramming to support glycolysis. However, the molecular mechanisms responsible for this change remain unclear. Here, we report that TGF-β1- or PDGF-induced CAFs switch from oxidative phosphorylation to aerobic glycolysis. We identify downregulation of isocitrate dehydrogenase 3α (IDH3α) as a marker for this switch. Furthermore, miR-424 downregulates IDH3α during CAF formation. Downregulation of IDH3α decreases the effective level ofα-ketoglutarate (α-KG) by reducing the ratio of α-KG to fumarate and succinate, resulting in PHD2 inhibition and HIF-1α protein stabilization. The accumulation of HIF-1α, in turn, promotes glycolysis by increasing the uptake of glucose, upregulating expression of glycolytic enzymes under normoxic conditions, and inhibiting oxidative phosphorylation by upregulating NDUFA4L2. CAFs from tumor samples exhibit low levels of IDH3α, and overexpression of IDH3α prevents transformation of fibroblasts into CAFs. Our studies reveal IDH3α to be a critical metabolic switch in CAFs.

MiR-186 regulates glycolysis through Glut1 during the formation of cancer-associated fibroblasts.

Emerging evidence has suggested that glycolysis is enhanced in cancer-associated fibroblasts (CAF), and miR-186 is downregulated during the CAF formation. However, it is not clear whether miR-186 is involved in the regulation of glycolysis and what the role of miR-186 plays during the CAF formation. In this study, quantitative PCR analysises show miR-186 is downregulated during the CAF formation. Moreover, miR-186 targets the 3' UTR of Glut1, and its overexpression results in the degradation of Glut1 mRNA, which eventually reduces the level of Glut1 protein. On the other hand, knockdown of miR-186 increased the expression of Glut1. Both time course and dose response experiments also demonstrated that the protein and mRNA levels of Glut1 increase during CAF formation, according to Western blot and quantitative PCR analyses, respectively. Most importantly, besides the regulation on cell cycle progression, miR-186 regulates glucose uptake and lactate production which is mediated by Glut1. These observations suggest that miR-186 plays important roles in glycolysis regulation as well as cell cycle checkpoint activation.