Autologous Platelet-Rich Growth Factor Reduces M1 Macrophages and Modulates Inflammatory Microenvironments to Promote Sciatic Nerve Regeneration.

The failure of peripheral nerve regeneration is often associated with the inability to generate a permissive molecular and cellular microenvironment for nerve repair. Autologous therapies, such as platelet-rich plasma (PRP) or its derivative platelet-rich growth factors (PRGF), may improve peripheral nerve regeneration via unknown mechanistic roles and actions in macrophage polarization. In the current study, we hypothesize that excessive and prolonged inflammation might result in the failure of pro-inflammatory M1 macrophage transit to anti-inflammatory M2 macrophages in large nerve defects. PRGF was used in vitro at the time the unpolarized macrophages (M0) macrophages were induced to M1 macrophages to observe if PRGF altered the secretion of cytokines and resulted in a phenotypic change. PRGF was also employed in the nerve conduit of a rat sciatic nerve transection model to identify alterations in macrophages that might influence excessive inflammation and nerve regeneration. PRGF administration reduced the mRNA expression of tumor necrosis factor-α (TNFα), interleukin-1ß (IL-1ß), and IL-6 in M0 macrophages. Increased CD206 substantiated the shift of pro-inflammatory cytokines to the M2 regenerative macrophage. Administration of PRGF in the nerve conduit after rat sciatic nerve transection promoted nerve regeneration by improving nerve gross morphology and its targeted gastrocnemius muscle mass. The regenerative markers were increased for regrown axons (protein gene product, PGP9.5), Schwann cells (S100ß), and myelin basic protein (MBP) after 6 weeks of injury. The decreased expression of TNFα, IL-1ß, IL-6, and CD68+ M1 macrophages indicated that the inflammatory microenvironments were reduced in the PRGF-treated nerve tissue. The increase in RECA-positive cells suggested the PRGF also promoted angiogenesis during nerve regeneration. Taken together, these results indicate the potential role and clinical implication of autologous PRGF in regulating inflammatory microenvironments via macrophage polarization after nerve transection.

CD28 engagement inhibits CD73-mediated regulatory activity of CD8+ T cells.

CD28 is required for T cell activation as well as the generation of CD4+Foxp3+ Treg. It is unclear, however, how CD28 costimulation affects the development of CD8+ T cell suppressive function. Here, by use of Hepa1.6.gp33 in vitro killing assay and B16.gp33 tumor mouse model we demonstrate that CD28 engagement during TCR ligation prevents CD8+ T cells from becoming suppressive. Interestingly, our results showed that ectonucleotidase CD73 expression on CD8+ T cells is upregulated in the absence of CD28 costimulation. In both murine and human tumor-bearing hosts, CD73 is upregulated on CD28-CD8+ T cells that infiltrate the solid tumor. UPLC-MS/MS analysis revealed that CD8+ T cells activation without CD28 costimulation produces elevated levels of adenosine and that CD73 mediates its production. Adenosine receptor antagonists block CD73-mediated suppression. Our data support the notion that CD28 costimulation inhibits CD73 upregulation and thereby prevents CD8+ T cells from becoming suppressive. This study uncovers a previously unidentified role for CD28 costimulation in CD8+ T cell activation and suggests that the CD28 costimulatory pathway can be a potential target for cancer immunotherapy.

miR-524-5p reduces the progression of the BRAF inhibitor-resistant melanoma.

BRAF inhibitors were approved for the treatment of BRAF-mutant melanoma. However, most patients acquire the resistance to BRAF inhibitors after several months of treatment. miR-524-5p is considered as a tumor suppressor in many cancers, including melanoma. In this study, we investigated the biological functions of miR-524-5p in melanoma with acquired resistance to BRAF inhibitor and evaluated the endogenous miR-524-5p expression as a biomarker for melanoma. The results showed that the expression of miR-524-5p was 0.481-fold lower in melanoma tissues (n = 117) than in nevus tissues (n = 40). Overexpression of miR-524-5p significantly reduced proliferative, anchorage-independent growth, migratory and invasive abilities of BRAF inhibitor-resistant melanoma cells. Moreover, the introduction of miR-524-5p led to a reduced development of BRAF inhibitor-resistant melanoma in vivo. Remarkably, the MAPK/ERK signaling pathway was decreased after treatment with miR-524-5p. Furthermore, next-generation sequencing analysis implied that the complement system, leukocyte extravasation, liver X receptor/retinoid-X-receptor activation, and cAMP-mediated signaling may be related to miR-524-5p-induced pathways in the resistant cells. The miR-524-5p level was higher on average in complete response and long-term partial response patients than in progressive disease and short-term partial response patients treated with BRAF inhibitors. Our results proposed that miR-524-5p could be considered as a target for treatment BRAF inhibitor-resistant melanoma and a prognostic marker in the response of patients to BRAF inhibitors for melanoma.

Selection of a malignant subpopulation from a colorectal cancer cell line.

Colorectal cancer (CRC) is a leading cause of cancer-associated mortality worldwide; therefore, there is an emerging need for novel experimental models that allow for the identification and validation of biomarkers for CRC-specific progression. In the present study, a repeated sphere-forming assay was used as a strategy to select a malignant subpopulation from a CRC cell line, namely HCT116. The assay was validated by confirming that canonical stemness markers were upregulated in the sphere state at every generation of the selection assay. The resulting subpopulation, after eight rounds of selection, exhibited increased sphere-forming capacity in vitro and increased tumorigenicity in vivo. Furthermore, dipeptidase 1 (DPEP1) was identified as the major differentially expressed gene in the selected clone, and its depletion suppressed the elevated sphere-forming capacity in vitro and tumorigenicity in vivo. Overall, the present study established an experimental strategy to isolate a malignant subpopulation from a CRC cell line. Additionally, results from the present model revealed that DPEP1 may serve as a promising prognostic biomarker for CRC.

Understanding Breast Implant Illness, Before and After Explantation: A Patient-Reported Outcomes Study.

BACKGROUND: Breast implant illness (BII) after aesthetic breast augmentation remains a poorly defined syndrome encompassing a wide spectrum of symptoms. While previously published series have observed overall symptomatic improvement after breast implant removal, there is a lack of studies evaluating changes in specific symptoms over time. The purpose of this study was to gain an understanding of symptoms associated with BII, and to evaluate how these symptoms change after removal of breast implants and total capsulectomy (explantation). We hypothesized that patients presenting with BII would experience both immediate and sustained improvement in constitutional symptoms after explantation. METHODS: A retrospective study of all patients who underwent explantation by a single surgeon over 2 years was conducted. Repeated-measures analysis of variance accounting for dependency was used to compare symptoms before and after surgery. Multivariate analyses and linear regression models were used to examine the impact of patient- and implant-related factors on changes in symptoms. RESULTS: Seven hundred fifty patients met inclusion criteria. Mean preoperative survey score (26.19 ± 11.24) was significantly different from mean postoperative survey score at less than 30 days (9.49 ± 7.56) and greater than 30 days (9.46 ± 7.82, P < 0.001). Patients with a BMI greater than 30 or those with clinically detectable contracture on examination showed greater improvement on their survey scores (P = 0.039, 0.034, respectively). CONCLUSIONS: Although BII encompasses a large range of symptoms, subjects in this study demonstrated significant and sustained improvement in 11 common symptom domains. This improvement was demonstrable within the first 30 days postoperatively and was maintained beyond 30 days. The study demonstrated a strong association of explantation and specific symptom improvement within the patient population studied. Future investigation will further elucidate possible biologic phenomena to better characterize the pathophysiology and mechanism of BII.

The extracellular domain of epithelial cell adhesion molecule (EpCAM) enhances multipotency of mesenchymal stem cells through EGFR-LIN28-LET7 signaling.

Mesenchymal stem cells (MSCs) are widely considered to be an attractive cell source for regenerative therapies, but maintaining multipotency and self-renewal in cultured MSCs is especially challenging. Hence, the development and mechanistic description of strategies that help promote multipotency in MSCs will be vital to future clinical use. Here, using an array of techniques and approaches, including cell biology, RT-quantitative PCR, immunoblotting, immunofluorescence, flow cytometry, and ChIP assays, we show that the extracellular domain of epithelial cell adhesion molecule (EpCAM) (EpEX) significantly increases the levels of pluripotency factors through a signaling cascade that includes epidermal growth factor receptor (EGFR), signal transducer and activator of transcription 3 (STAT3), and Lin-28 homolog A (LIN28) and enhances the proliferation of human bone marrow MSCs. Moreover, we found that EpEX-induced LIN28 expression reduces the expression of the microRNA LET7 and up-regulates that of the transcription factor high-mobility group AT-hook 2 (HMGA2), which activates the transcription of pluripotency factors. Surprisingly, we found that EpEX treatment also enhances osteogenesis of MSCs under differentiation conditions, as evidenced by increases in osteogenic markers, including Runt-related transcription factor 2 (RUNX2). Taken together, our results indicate that EpEX stimulates EGFR signaling and thereby context-dependently controls MSC states and activities, promoting cell proliferation and multipotency under maintenance conditions and osteogenesis under differentiation conditions.

miR-596 Modulates Melanoma Growth by Regulating Cell Survival and Death.

Tumors grow because cancer cells lack the ability to balance cell survival and death signaling pathways. miR-596, a microRNA located at the 8p23.3 locus, has been shown by the TCGA-Assembler to be deleted in a significant number of melanoma samples. Here, we also validated the low levels of miR-596 in melanoma compared to tissue nevi, and Kaplan-Meier curve analysis revealed that low miR-596 expression was associated with worse overall survival. Moreover, we showed that miR-596 overexpression effectively inhibited MAPK/ERK signaling, cell proliferation, migration, and invasion and increased the cell apoptosis of melanoma cells. In addition, we found that miR-596 directly targets MEK1 and two apoptotic proteins, MCL1, and BCL2L1, in melanoma cells. Our findings indicated that miR-596 is an important miRNA that both negatively regulates the MAPK/ERK signaling pathway by targeting MEK1 and modulates the apoptosis pathway by targeting MCL1 and BCL2L1, suggesting that miR-596 could be a therapeutic candidate for treating melanoma, and a prognostic factor for melanoma patients.

PARP1 controls KLF4-mediated telomerase expression in stem cells and cancer cells.

Telomerase is highly expressed in cancer and embryonic stem cells (ESCs) and implicated in controlling genome integrity, cancer formation and stemness. Previous studies identified that Krüppel-like transcription factor 4 (KLF4) activates telomerase reverse transcriptase (TERT) expression and contributes to the maintenance of self-renewal in ESCs. However, little is known about how KLF4 regulates TERT expression. Here, we discover poly(ADP-ribose) polymerase 1 (PARP1) as a novel KLF4-interacting partner. Knockdown of PARP1 reduces TERT expression and telomerase activity not only in cancer cells, but also in human and mouse ESCs. Recruitment of KLF4 to TERT promoter is reduced in PARP1-suppressed cells. The poly(ADP-ribose) polymerase activity is dispensable, while the oligo(ADP-ribose) polymerase activity is required for the PARP1- and KLF4-mediated TERT activation. Repression of Parp1 in mouse ESCs decreases expression of pluripotent markers and induces differentiation. These results suggest that PARP1 recruits KLF4 to activate telomerase expression and stem cell pluripotency, indicating a positive regulatory role of the PARP1-KLF4 complex in telomerase expression in cancer and stem cells.

CHAC2 is essential for self-renewal and glutathione maintenance in human embryonic stem cells.

Glutathione (GSH), the major non-enzymatic antioxidant, plays a critical role in cellular reactive oxygen species (ROS) neutralization. Moreover, GSH is required for the self-renewal maintenance of human embryonic stem cells (hESCs), and is highly accumulated in undifferentiated cells. Among 8 GSH biosynthesis-related enzymes, we found CHAC2 is highly enriched in undifferentiated hESCs. CHAC2 downregulation in hESCs efficiently decreased the levels of GSH and blocked self-renewal. The self-renewal of sh-CHAC2 cells can be rescued by GSH supplement. CHAC2 downregulation promoted mesoderm differentiation and hampered both teratoma formation and the expression of Nrf2 and glutamate-cysteine ligase (GCL). Notably, CHAC1 knockdown restored the self-renewability of CHAC2-downregulated cells. Although both CHAC1 and CHAC2 purified protein alone showed the catalytic activities to GSH, our data extraordinarily revealed that CHAC2 prevented CHAC1-mediated GSH degradation, which suggests that CHAC2 competes with CHAC1 to maintain GSH homeostasis. This is the first report to demonstrate that CHAC2 is critical for GSH maintenance and the novel roles of the CHAC family in hESC renewal.

Elimination of undifferentiated human embryonic stem cells by cardiac glycosides.

An important safety concern in the use of human pluripotent stem cells (hPSCs) is tumorigenic risk, because these cells can form teratomas after an in vivo injection at ectopic sites. Several thousands of undifferentiated hPSCs are sufficient to induce teratomas in a mouse model. Thus, it is critical to remove all residue-undifferentiated hPSCs that have teratoma potential before the clinical application of hPSC-derived cells. In this study, our data demonstrated the cytotoxic effects of cardiac glycosides, such as digoxin, lanatoside C, bufalin, and proscillaridin A, in human embryonic stem cells (hESCs). This phenomenon was not observed in human bone marrow mesenchymal stem cells (hBMMSCs). Most importantly, digoxin and lanatoside C did not affect the stem cells' differentiation ability. Consistently, the viability of the hESC-derived MSCs, neurons, and endothelium cells was not affected by the digoxin and lanatoside C treatment. Furthermore, the in vivo experiments demonstrated that digoxin and lanatoside C prevented teratoma formation. To the best of our knowledge, this study is the first to describe the cytotoxicity and tumor prevention effects of cardiac glycosides in hESCs. Digoxin and lanatoside C are also the first FDA-approved drugs that demonstrated cytotoxicity in undifferentiated hESCs.

Efficient Generation of Chemically Induced Mesenchymal Stem Cells from Human Dermal Fibroblasts.

Human mesenchymal stromal/stem cells (MSCs) are multipotent and currently undergoing hundreds of clinical trials for disease treatments. To date, no studies have generated induced MSCs from skin fibroblasts with chemicals or growth factors. Here, we established the first chemical method to convert primary human dermal fibroblasts into multipotent, induced MSC-like cells (iMSCs). The conversion method uses a defined cocktail of small molecules and growth factors, and it can achieve efficient conversion with an average rate of 38% in 6 days. The iMSCs have much higher clonogenicity than fibroblasts, and they can be maintained and expanded in regular MSC medium for at least 8 passages and further differentiated into osteoblasts, adipocytes, and chondrocytes. Moreover, the iMSCs can suppress LPS-mediated acute lung injury as effectively as bone marrow-derived mesenchymal stem cells. This finding may greatly benefit stem cell biology, cell therapy, and regenerative medicine.

EpEX/EpCAM and Oct4 or Klf4 alone are sufficient to generate induced pluripotent stem cells through STAT3 and HIF2α.

Epithelial cell adhesion molecule (EpCAM) was reported to be cleaved into extracellular domain of EpCAM (EpEX) and intracellular domain of EpCAM (EpICD). We previously reported that EpCAM serves as a potent stem cell marker which is highly and selectively expressed by undifferentiated rather than differentiated hESC. However, the functional role of EpCAM remains elusive. Here, we found that EpEX and EpCAM enhance the efficiency of OSKM reprogramming. Interestingly, Oct4 or Klf4 alone, but not Sox2, can successfully reprogram fibroblasts into iPSCs with EpEX and EpCAM. Moreover, EpEX and EpCAM trigger reprogramming via activation of STAT3, which leads to the nuclear-translocation of HIF2α. This study reveals the importance of a novel EpEX/EpCAM-STAT3-HIF2α signal in the reprogramming process, and uncovers a new means of triggering reprogramming by delivery of soluble and transmembrane proteins.

Derivation of Patient Specific Pluripotent Stem Cells Using Clinically Discarded Cumulus Cells.

Induced pluripotent stem cells (iPSCs) are powerful tools for basic and translational research, as well as regenerative medicine. In routine human in vitro fertilization (IVF) practices, cumulus cells (CCs) are discarded, representing a potential source of biological materials for regenerative medicine. In this study, we derived patient-specific iPSCs using CCs from human infertility clinics for the first time. The human cumulus cell derived iPSCs (hc-iPSCs) were characterized for growth, karyotype, expression of pluripotency genes, and were subjected to embryoid bodies (EBs) and teratoma assays to evaluate their differentiation capacity. Hc-iPSCs display typical iPSC characteristics, and are capable of differentiating into all germ layers in vitro and in vivo. We further show that putative primordial germ cell like cells (PGCLCs) can be derived using hc-iPSCs. Our data demonstrate the feasibility of deriving patient-specific pluripotent stem cells using CCs.

Regulation of EBV LMP1-triggered EphA4 downregulation in EBV-associated B lymphoma and its impact on patients' survival.

Epstein-Barr virus (EBV), an oncogenic human virus, is associated with several lymphoproliferative disorders, including Burkitt lymphoma, Hodgkin disease, diffuse large B-cell lymphoma (DLBCL), and posttransplant lymphoproliferative disorder (PTLD). In vitro, EBV transforms primary B cells into lymphoblastoid cell lines (LCLs). Recently, several studies have shown that receptor tyrosine kinases (RTKs) play important roles in EBV-associated neoplasia. However, details of the involvement of RTKs in EBV-regulated B-cell neoplasia and malignancies remain largely unclear. Here, we found that erythropoietin-producing hepatocellular receptor A4 (EphA4), which belongs to the largest RTK Eph family, was downregulated in primary B cells post-EBV infection at the transcriptional and translational levels. Overexpression and knockdown experiments confirmed that EBV-encoded latent membrane protein 1 (LMP1) was responsible for this EphA4 suppression. Mechanistically, LMP1 triggered the extracellular signal-regulated kinase (ERK) pathway and promoted Sp1 to suppress EphA4 promoter activity. Functionally, overexpression of EphA4 prevented LCLs from proliferation. Pathologically, the expression of EphA4 was detected in EBV(-) tonsils but not in EBV(+) PTLD. In addition, an inverse correlation of EphA4 expression and EBV presence was verified by immunochemical staining of EBV(+) and EBV(-) DLBCL, suggesting EBV infection was associated with reduced EphA4 expression. Analysis of a public data set showed that lower EphA4 expression was correlated with a poor survival rate of DLBCL patients. Our findings provide a novel mechanism by which EphA4 can be regulated by an oncogenic LMP1 protein and explore its possible function in B cells. The results provide new insights into the role of EphA4 in EBV(+) PTLD and DLBCL.

GIT1 promotes lung cancer cell metastasis through modulating Rac1/Cdc42 activity and is associated with poor prognosis.

G-protein-coupled receptor kinase interacting protein 1 (GIT1) is participated in cell movement activation, which is a fundamental process during tissue development and cancer progression. GIT1/PIX forming a functional protein complex that contributes to Rac1/Cdc42 activation, resulting in increasing cell mobility. Although the importance of Rac1/Cdc42 activation is well documented in cancer aggressiveness, the clinical importance of GIT1 remains largely unknown. Here, we investigated the clinical significance of GIT1 expression in non-small-cell lung cancer (NSCLC) and also verified the importance of GIT1-Rac1/Cdc42 axis in stimulating NSCLC cell mobility. The result indicated higher GIT1 expression patients had significantly poorer prognoses in disease-free survival (DFS) and overall survival (OS) compared with lower GIT1 expression patients. Higher GIT1 expression was an independent prognostic factor by multivariate analysis and associated with migration/invasion of NSCLC cells in transwell assay. In vivo studies indicated that GIT1 promotes metastasis of NSCLC cells. Finally, GIT1 was found to stimulate migration/invasion by altering the activity of Rac1/Cdc42 in NSCLC cells. Together, the GIT1 expression is associated with poor prognosis in patients with NSCLC. GIT1 is critical for the invasiveness of NSCLC cells through stimulating the activity of Rac1/Cdc42.

RBMY, a novel inhibitor of glycogen synthase kinase 3ß, increases tumor stemness and predicts poor prognosis of hepatocellular carcinoma.

UNLABELLED: Male predominance of hepatocellular carcinoma (HCC) occurs particularly among young children aged 6-9 years, indicative of a possible role of the Y chromosome-encoded oncogene in addition to an androgenic effect. The discovery of oncogenic activation of RBMY (RNA-binding motif on Y chromosome), which is absent in normal hepatocytes but present in male HCC tissues, sheds light on this issue. Herein, we report on a critical hepatocarcinogenic role of RBMY and its ontogenic origin. During liver development, the Ser/Thr phosphorylated RBMY is expressed in the cytoplasm of human and rodent fetal livers. It is then silenced in mature hepatocytes and restricted to scarce expression in the bile ductular cells. Upon hepatocarcinogenesis, a noteworthy increase of cytoplasmic and nuclear RBMY is observed in HCC tissues; however, only the former is expressed dominantly in hepatic cancer stem cells and correlates significantly to a poor prognosis and decreased survival rate in HCC patients. Cytoplasmic expression of RBMY, which is mediated by binding to nuclear exporter chromosome region maintenance 1 and further enriched upon Wnt-3a stimulation, confers upon tumor cells the traits of cancer stem cell by augmenting self-renewal, chemoresistance, cell-cycle progression, proliferation, and xenograft tumor growth. This is achieved mechanistically through increasing Ser9 phosphorylation-inactivation of glycogen synthase kinase 3ß by RBMY, thereby impeding the glycogen synthase kinase 3ß-dependent degradation of ß-catenin and eventually inducing the nuclear entry of ß-catenin for the transcription of downstream oncogenes. CONCLUSION: RBMY is a novel oncofetal protein that plays a key role in attenuating glycogen synthase kinase 3ß activity, leading to aberrant activation of Wnt/ß-catenin signaling, which facilitates malignant hepatic stemness; because of its absence from normal human tissues except the testis, RBMY represents a feasible therapeutic target for the selective eradication of HCC cells in male patients.

Akt suppresses DLK for maintaining self-renewal of mouse embryonic stem cells.

Mouse embryonic stem cells (ES cells) can proliferate indefinitely. To identify potential signals involved in suppression of self-renewal, we previously screened a kinase/phosphatase expression library in ES cells, and observed that inhibition of Dual Leucine zipper-bearing Kinase (DLK) increased relative cell numbers. DLK protein was detected in both the pluripotent and differentiated states of mouse ES cells while DLK kinase activity increased upon differentiation. Overexpression of DLK in mouse ES cells displayed reductions in relative cell/colony numbers and Nanog expression, suggesting a suppressive role of DLK in self-renewal. By examining protein sequences of DLK, we identified 2 putative Akt phosphorylation sites at S584 and T659. Blocking PI3K/Akt signaling with LY-294002 enhanced DLK kinase activity dramatically. We found that Akt interacts with and phosphorylates DLK. Mutations of DLK amino acid residues at putative Akt phosphorylation sites (S584A, T659A, or S584A and T659A) diminished the level of DLK phosphorylation. While the mutated DLKs (S584A, T659A, or S584A and T659A) were expressed, a further reduction in cell/colony numbers and Nanog expression appeared in mouse ES cells. In addition, these mutant DLKs (S584A, T659A, or S584A and T659A) exhibited more robust kinase activity and cell death compared to wild type DLK or green fluorescence (GFP) controls. In summary, our results show that DLK functions to suppress self-renewal of mouse ES cells and is restrained by Akt phosphorylation.

miR-524-5p suppresses the growth of oncogenic BRAF melanoma by targeting BRAF and ERK2.

It has been well documented that miRNAs can modulate the effectiveness of cancer-associated signaling pathways. Mitogen-activated protein kinase (MAPK/ERK) signaling plays an essential role in the progression of many cancers, including melanoma and colon cancers. However, no single miRNA is reported to directly target multiple components of the MAPK/ERK pathway. We performed a miRNA PCR array screening with various MAPK/ERK signaling activities. The miRNA array data revealed that the expression of miR-524-5p was decreased in cells with an active MAPK/ERK pathway and confirmed that the expression of miR-524-5p is inversely associated with the activity of the MAPK/ERK pathway. We demonstrated that miR-524-5p directly binds to the 3'-untranslated regions of both BRAFandERK2 and suppresses the expression of these proteins. Because BRAF and ERK2 are the main components of MAPK signaling, the overexpression of miR-524-5p effectively inhibits MAPK/ERK signaling, tumor proliferation, and melanoma cell migration. Moreover, tumors overexpressing miR-524-5p were significantly smaller than those of the negative control mice. Our findings provide new insight into the role of miR-524-5p as an important miRNA that negatively regulates the MAPK/ERK signaling pathway, suggesting that miR-524-5p could be a potent therapeutic candidate for melanoma treatment.

Squalene synthase induces tumor necrosis factor receptor 1 enrichment in lipid rafts to promote lung cancer metastasis.

RATIONALE: Metabolic alterations contribute to cancer development and progression. However, the molecular mechanisms relating metabolism to cancer metastasis remain largely unknown. OBJECTIVES: To identify a key metabolic enzyme that is aberrantly overexpressed in invasive lung cancer cells and to investigate its functional role and prognostic value in lung cancer. METHODS: The differential expression of metabolic enzymes in noninvasive CL1-0 cells and invasive CL1-5 cells was analyzed by a gene expression microarray. The expression of target genes in clinical specimens from patients with lung cancer was examined by immunohistochemistry. Pharmacologic and gene knockdown/overexpression approaches were used to investigate the function of the target gene during invasion and metastasis in vitro and in vivo. The association between the target gene expression and clinicopathologic parameters was further analyzed. Bioinformatic analyses were used to discover the signaling pathways involved in target gene-regulated invasion and migration. MEASUREMENTS AND MAIN RESULTS: Squalene synthase (SQS) was up-regulated in CL1-5 cells and in the tumor regions of the lung cancer specimens. Loss of function or knockdown of SQS significantly inhibited invasion/migration and metastasis in cell and animal models and vice versa. High expression of SQS was significantly associated with poor prognosis among patients with lung cancer. Mechanistically, SQS contributed to a lipid-raft-localized enrichment of tumor necrosis factor receptor 1 in a cholesterol-dependent manner, which resulted in the enhancement of nuclear factor-κB activation leading to matrix metallopeptidase 1 up-regulation. CONCLUSIONS: Up-regulation of SQS promotes metastasis of lung cancer by enhancing tumor necrosis factor-α receptor 1 and nuclear factor-κB activation and matrix metallopeptidase 1 expression. Targeting SQS may have considerable potential as a novel therapeutic strategy to treat metastatic lung cancer.