NF-κB Activation In Fibroblasts Research Articles

Sam68 impedes the recovery of arterial injury by augmenting inflammatory response

ObjectiveThe role of Src-associated-in-mitosis-68-kDa (Sam68) in cardiovascular biology has not been studied. A recent report suggests that Sam68 promotes TNF-α–induced NF-κB activation in fibroblasts. Here we sought to dissect the molecular mechanism by which Sam68 regulates NF-κB signaling and its functional significance in vascular injury. Approach and resultsThe endothelial denudation injury was induced in the carotid artery of Sam68-null (Sam68−/−) and WT mice. Sam68−/− mice displayed an accelerated re-endothelialization and attenuated neointima hyperplasia, which was associated with a reduced macrophage infiltration and lowered expression of pro-inflammatory cytokines in the injured vessels. Remarkably, the ameliorated vascular remodeling was recapitulated in WT mice after receiving transplantation of bone marrow (BM) from Sam68−/− mice, suggesting the effect was attributable to BM-derived inflammatory cells. In cultured Raw264.7 macrophages, knockdown of Sam68 resulted in a significant reduction in the TNF-α–induced expression of TNF-α, IL-1β, and IL-6 and in the level of nuclear phospho-p65, indicating attenuated NF-κB activation; and these results were confirmed in peritoneal and BM-derived macrophages of Sam68−/− vs. WT mice. Furthermore, co-immunoprecipitation and mass-spectrometry identified Filamin A (FLNA) as a novel Sam68-interacting protein upon TNF-α treatment. Loss- and gain-of-function experiments suggest that Sam68 and FLNA are mutually dependent for NF-κB activation and pro-inflammatory cytokine expression, and that the N-terminus of Sam68 is required for TRAF2-FLNA interaction. ConclusionsSam68 promotes pro-inflammatory response in injured arteries and impedes recovery by interacting with FLNA to stabilize TRAF2 on the cytoskeleton and consequently potentiate NF-κB signaling.

NF‐κB is activated in oesophageal fibroblasts in response to a paracrine signal generated by acid‐exposed primary oesophageal squamous cells

Oesophageal exposure to duodenogastro-oesophageal refluxate leads to reflux oesophagitis and is implicated in the development of Barrett's metaplasia (BM). NF-κB signalling in epithelial cells is associated with the activation of transcription factors believed to be central to BM development, whilst NF-κB activation in fibroblasts plays a critical role in matrix remodelling. Our aim was to study the effects of acid exposure on NF-κB activation in primary human oesophageal fibroblasts (HOFs) and primary and immortalized oesophageal squames and to investigate any epithelial/stromal interactions in the response of these cells to acid. Primary HOFs and primary and immortalized oesophageal epithelial cells were exposed to acid (pH 7 - pH 4 ≤ 120 min) in single or pulsed treatments. Conditioned medium from epithelial cells following acid exposure was also applied to fibroblasts. Cell viability was determined by MTT-ESTA. NF-κB activation was determined by cellular localization of NF-κB/p65 visualized by immunofluorescence. Conditioned medium from oesophageal epithelial cells, subjected to pH 5 pulsatile exposure, activated NF-κB in fibroblasts, with some inter-patient variability, but these conditions did not directly activate NF-κB in the epithelial cells themselves. Significant NF-κB activation was seen in the epithelial cells but only with greater acidity and exposure times (pH 4, 60-120 min). Our findings show that acid exposure can cause indirect activation of stromal cells by epithelial-stromal interactions. This may contribute to the pathogenesis of oesophageal diseases, and the inter-patient variability may go some way to explain why some patients with reflux oesophagitis develop BM and others do not.

HIF1-alpha functions as a tumor promoter in cancer-associated fibroblasts, and as a tumor suppressor in breast cancer cells

Our recent studies have mechanistically implicated a loss of stromal Cav-1 expression and HIF1-alpha-activation in driving the cancer-associated fibroblast phenotype, through the paracrine production of nutrients via autophagy and aerobic glycolysis. However, it remains unknown if HIF1a-activation is sufficient to confer the cancer-associated fibroblast phenotype. To test this hypothesis directly, we stably-expressed activated HIF1a in fibroblasts and then examined their ability to promote tumor growth using a xenograft model employing human breast cancer cells (MDA-MB-231). Fibroblasts harboring activated HIF1a showed a dramatic reduction in Cav-1 levels and a shift towards aerobic glycolysis, as evidenced by a loss of mitochondrial activity, and an increase in lactate production. Activated HIF1a also induced BNIP3 and BNIP3L expression, markers for the autophagic destruction of mitochondria. Most importantly, fibroblasts expressing activated HIF1a increased tumor mass by ~2-fold and tumor volume by ~3-fold, without a significant increase in tumor angiogenesis. In this context, HIF1a also induced an increase in the lymph node metastasis of cancer cells. Similar results were obtained by driving NFκB activation in fibroblasts, another inducer of autophagy. Thus, activated HIF1a is sufficient to functionally confer the cancer-associated fibroblast phenotype. It is also known that HIF1a expression is required for the induction of autophagy in cancer cells. As such, we next directly expressed activated HIF1a in MDA-MB-231 cells and assessed its effect on tumor growth via xenograft analysis. Surprisingly, activated HIF1a in cancer cells dramatically suppressed tumor growth, resulting in a 2-fold reduction in tumor mass and a 3-fold reduction in tumor volume. We conclude that HIF1a activation in different cell types can either promote or repress tumorigenesis. Based on these studies, we suggest that autophagy in cancer-associated fibroblasts promotes tumor growth via the paracrine production of recycled nutrients, which can directly "feed" cancer cells. Conversely, autophagy in cancer cells represses tumor growth via their "self-digestion." Thus, we should consider that the activities of various known oncogenes and tumor-suppressors may be compartment and cell-type specific, and are not necessarily an intrinsic property of the molecule itself. As such, other "classic" oncogenes and tumor suppressors will have to be re-evaluated to determine their compartment specific effects on tumor growth and metastasis. Lastly, our results provide direct experimental support for the recently proposed "Autophagic Tumor Stroma Model of Cancer."

Autophagy in cancer associated fibroblasts promotes tumor cell survival

Recently, using a co-culture system, we demonstrated that MCF7 epithelial cancer cells induce oxidative stress in adjacent cancer-associated fibroblasts, resulting in the autophagic/lysosomal degradation of stromal caveolin-1 (Cav-1). However, the detailed signaling mechanism(s) underlying this process remain largely unknown. Here, we show that hypoxia is sufficient to induce the autophagic degradation of Cav-1 in stromal fibroblasts, which is blocked by the lysosomal inhibitor chloroquine. Concomitant with the hypoxia-induced degradation of Cav-1, we see the upregulation of a number of well-established autophagy/mitophagy markers, namely LC3, ATG16L, BNIP3, BNIP3L, HIF-1α and NFκB. In addition, pharmacological activation of HIF-1α drives Cav-1 degradation, while pharmacological inactivation of HIF-1 prevents the downregulation of Cav-1. Similarly, pharmacological inactivation of NFκB-another inducer of autophagy-prevents Cav-1 degradation. Moreover, treatment with an inhibitor of glutathione synthase, namely BSO, which induces oxidative stress via depletion of the reduced glutathione pool, is sufficient to induce the autophagic degradation of Cav-1. Thus, it appears that oxidative stress mediated induction of HIF1- and NFκB-activation in fibroblasts drives the autophagic degradation of Cav-1. In direct support of this hypothesis, we show that MCF7 cancer cells activate HIF-1α- and NFκB-driven luciferase reporters in adjacent cancer-associated fibroblasts, via a paracrine mechanism. Consistent with these findings, acute knock-down of Cav-1 in stromal fibroblasts, using an siRNA approach, is indeed sufficient to induce autophagy, with the upregulation of both lysosomal and mitophagy markers. How does the loss of stromal Cav-1 and the induction of stromal autophagy affect cancer cell survival? Interestingly, we show that a loss of Cav-1 in stromal fibroblasts protects adjacent cancer cells against apoptotic cell death. Thus, autophagic cancer-associated fibroblasts, in addition to providing recycled nutrients for cancer cell metabolism, also play a protective role in preventing the death of adjacent epithelial cancer cells. We demonstrate that cancer-associated fibroblasts upregulate the expression of TIGAR in adjacent epithelial cancer cells, thereby conferring resistance to apoptosis and autophagy. Finally, the mammary fat pads derived from Cav-1 (-/-) null mice show a hypoxia-like response in vivo, with the upregulation of autophagy markers, such as LC3 and BNIP3L. Taken together, our results provide direct support for the "Autophagic Tumor Stroma Model of Cancer Metabolism," and explain the exceptional prognostic value of a loss of stromal Cav-1 in cancer patients. Thus, a loss of stromal fibroblast Cav-1 is a biomarker for chronic hypoxia, oxidative stress and autophagy in the tumor microenvironment, consistent with its ability to predict early tumor recurrence, lymph node metastasis and tamoxifen-resistance in human breast cancers. Our results imply that cancer patients lacking stromal Cav-1 should benefit from HIF-inhibitors, NFκB-inhibitors, anti-oxidant therapies, as well as autophagy/lysosomal inhibitors. These complementary targeted therapies could be administered either individually or in combination, to prevent the onset of autophagy in the tumor stromal compartment, which results in a "lethal" tumor microenvironment.

NF-kappaB activation and interleukin 6 production in fibroblasts by estrogen receptor-negative breast cancer cell-derived interleukin 1alpha.

Several angiogenic factors and extracellular matrix-degrading enzymes that promote invasion and metastasis of cancer are produced by stromal fibroblasts that surround cancer cells. The expression of genes that code for some of these proteins is regulated by the transcription factor NF-kappaB. In this report, we demonstrate that conditioned medium (CM) from estrogen receptor (ER)-negative but not ER-positive breast cancer cells induces NF-kappaB in fibroblasts. In contrast, CM from both ER-positive and ER-negative breast cancer cells induces NF-kappaB in macrophages and endothelial cells. NF-kappaB activation in fibroblasts was accompanied by induction of interleukin 6 (IL-6) and urokinase plasminogen activator (uPA), both of which promote angiogenesis and metastasis. A survey of cytokines known for their ability to induce NF-kappaB identified IL-1alpha as the factor responsible for NF-kappaB activation in fibroblasts. Analysis of primary breast carcinomas revealed the presence of IL-1alpha transcripts in majority of lymph node-positive breast cancers. These results along with the known role of IL-1alpha and IL-6 in osteoclast formation provide insight into the mechanism of metastasis and hypercalcemia in advanced breast cancers.