Active immunosurveillance in the tumor microenvironment of colorectal cancer is associated with low frequency tumor budding and improved outcome.

Tumor budding (single tumor cells or small tumor cell clusters) at the invasion front of colorectal cancer (CRC) is an adverse prognostic indicator linked to epithelial-mesenchymal transition. This study characterized the immunogenicity of tumor buds by analyzing the expression of the major histocompatibility complex (MHC) class I in the invasive tumor cell compartment. We hypothesized that maintenance of a functional MHC-I antigen presentation pathway, activation of CD8+ T-cells, and release of antitumoral effector molecules such as cytotoxic granule-associated RNA binding protein (TIA1) in the tumor microenvironment can counter tumor budding and favor prolonged patient outcome. Therefore, a well-characterized multipunch tissue microarray of 220 CRCs was profiled for MHC-I, CD8, and TIA1 by immunohistochemistry. Topographic expression analysis of MHC-I was performed using whole tissue sections (n = 100). Kirsten rat sarcoma viral oncogene homolog (KRAS) and B-Raf proto-oncogene, serine/threonine kinase (BRAF) mutations, mismatch repair (MMR) protein expression, and CpG-island methylator phenotype (CIMP) were investigated. Our results demonstrated that membranous MHC-I expression is frequently down-regulated in the process of invasion. Maintained MHC-I at the invasion front strongly predicted low-grade tumor budding (P = 0.0004). Triple-positive MHC-I/CD8/TIA1 in the tumor microenvironment predicted early T-stage (P = 0.0031), absence of lymph node metastasis (P = 0.0348), lymphatic (P = 0.0119) and venous invasion (P = 0.006), and highly favorable 5-year survival (90.9% vs 39.3% in triple-negative patients; P = 0.0032). MHC-I loss was frequent in KRAS-mutated, CD8+ CRC (P = 0.0228). No relationship was observed with CIMP, MMR, or BRAF mutation. In conclusion, tumor buds may evade immune recognition through downregulation of membranous MHC-I. A combined profile of MHC-I/CD8/TIA1 improves the prognostic value of antitumoral effector cells and should be preferred to a single marker approach.

Tumour border configuration in colorectal cancer: proposal for an alternative scoring system based on the percentage of infiltrating margin.

AIMS Information on tumour border configuration (TBC) in colorectal cancer (CRC) is currently not included in most pathology reports, owing to lack of reproducibility and/or established evaluation systems. The aim of this study was to investigate whether an alternative scoring system based on the percentage of the infiltrating component may represent a reliable method for assessing TBC. METHODS AND RESULTS Two hundred and fifteen CRCs with complete clinicopathological data were evaluated by two independent observers, both 'traditionally' by assigning the tumours into pushing/infiltrating/mixed categories, and alternatively by scoring the percentage of infiltrating margin. With the pushing/infiltrating/mixed pattern method, interobserver agreement (IOA) was moderate (κ = 0.58), whereas with the percentage of infiltrating margins method, IOA was excellent (intraclass correlation coefficient of 0.86). A higher percentage of infiltrating margin correlated with adverse features such as higher grade (P = 0.0025), higher pT (P = 0.0007), pN (P = 0.0001) and pM classification (P = 0.0063), high-grade tumour budding (P < 0.0001), lymphatic invasion (P < 0.0001), vascular invasion (P = 0.0032), and shorter survival (P = 0.0008), and was significantly associated with an increased probability of lymph node metastasis (P < 0.001). CONCLUSIONS Information on TBC gives additional prognostic value to pathology reports on CRC. The novel proposed scoring system, by using the percentage of infiltrating margin, outperforms the 'traditional' way of reporting TBC. Additionally, it is reproducible and simple to apply, and can therefore be easily integrated into daily diagnostic practice.

High tumor budding stratifies breast cancer with metastatic properties.

Tumor budding refers to single or small cluster of tumor cells detached from the main tumor mass. In colon cancer high tumor budding is associated with positive lymph nodes and worse prognosis. Therefore, we investigated the value of tumor budding as a predictive feature of lymph node status in breast cancer (BC). Whole tissue sections from 148 surgical resection specimens (SRS) and 99 matched preoperative core biopsies (CB) with invasive BC of no special type were analyzed on one slide stained with pan-cytokeratin. In SRS, the total number of intratumoral (ITB) and peripheral tumor buds (PTB) in ten high-power fields (HPF) were counted. A bud was defined as a single tumor cell or a cluster of up to five tumor cells. High tumor budding equated to scores averaging >4 tumor buds across 10HPFs. In CB high tumor budding was defined as ≥10 buds/HPF. The results were correlated with pathological parameters. In SRS high PTB stratified BC with lymph node metastases (p ≤ 0.03) and lymphatic invasion (p ≤ 0.015). In CB high tumor budding was significantly (p = 0.0063) associated with venous invasion. Pathologists are able, based on morphology, to categorize BC into a high and low risk groups based in part on lymph node status. This risk assessment can be easily performed during routine diagnostics and it is time and cost effective. These results suggest that high PTB is associated with loco-regional metastasis, highlighting the possibility that this tumor feature may help in therapeutic decision-making.

Expression of E-cadherin repressors SNAIL, ZEB1 and ZEB2 by tumour and stromal cells influences tumour-budding phenotype and suggests heterogeneity of stromal cells in pancreatic cancer.

BACKGROUND There is evidence that tumour-stroma interactions have a major role in the neoplastic progression of pancreatic ductal adenocarcinoma (PDAC). Tumour budding is thought to reflect the process of epithelial-mesenchymal transition (EMT); however, the relationship between tumour buds and EMT remains unclear. Here we characterize the tumour-budding- and stromal cells in PDAC at protein and mRNA levels concerning factors involved in EMT. METHODS mRNA in situ hybridisation and immunostaining for E-cadherin, β-catenin, SNAIL1, ZEB1, ZEB2, N-cadherin and TWIST1 were assessed in the main tumour, tumour buds and tumour stroma on multipunch tissue microarrays from 120 well-characterised PDACs and associated with the clinicopathological features, including peritumoural (PTB) and intratumoural (ITB) budding. RESULTS Tumour-budding cells showed increased levels of ZEB1 (P<0.0001) and ZEB2 (P=0.0119) and reduced E-cadherin and β-catenin (P<0.0001, each) compared with the main tumour. Loss of membranous β-catenin in the main tumour (P=0.0009) and tumour buds (P=0.0053), without nuclear translocation, as well as increased SNAIL1 in tumour and stromal cells (P=0.0002, each) correlated with high PTB. ZEB1 overexpression in the main tumour-budding and stromal cells was associated with high ITB (P=0.0084; 0.0250 and 0.0029, respectively) and high PTB (P=0.0005; 0.0392 and 0.0007, respectively). ZEB2 overexpression in stromal cells correlated with higher pT stage (P=0.03), lymphatic invasion (P=0.0172) and lymph node metastasis (P=0.0152). CONCLUSIONS In the tumour microenvironment of phenotypically aggressive PDAC, tumour-budding cells express EMT hallmarks at protein and mRNA levels underlining their EMT-type character and are surrounded by stromal cells expressing high levels of the E-cadherin repressors ZEB1, ZEB2 and SNAIL1, this being strongly associated with the tumour-budding phenotype. Moreover, our findings suggest the existence of subtypes of stromal cells in PDAC with phenotypical and functional heterogeneity.

Blood and lymphatic vessel invasion in pT1 colorectal cancer: an international concordance study

AIM This study was performed to evaluate the concordance in pathological assessments of blood and lymphatic vessel invasion (BLI) in pT1 colorectal cancers and to assess the effect of diagnostic criterion on consistency in the assessment of BLI. METHODS Forty consecutive patients undergoing surgical resection of pT1 colorectal cancers were entered into this study. H&E-stained, D2-40-stained and elastica-stained slides from the tumours were examined by 18 pathologists from seven countries. The 40 cases were divided into two cohorts with 20 cases each. In cohort 1, pathologists diagnosed BLI using criteria familiar to them; all Japanese pathologists used a criterion of BLI from the Japanese Society for Cancer of the Colon and Rectum (JSCCR). In cohort 2, all pathologists used the JSCCR diagnostic criterion. RESULTS In cohort 1, diagnostic concordance was moderate in the US/Canadian and European pathologists. There were no differences in the consistency compared with results for Japanese pathologists, and no improvement in the diagnostic concordance was found for using the JSCCR criterion. However, in cohort 2, the JSCCR criterion decreased the consistency of BLI diagnosis in the US/Canadian and European pathologists. The level of decreased consistency in the assessment of BLI was different between the US/Canadian and European pathologists. CONCLUSIONS A uniform criterion strongly influences the diagnostic consistency of BLI but may not always improve the concordance. Further study is required to achieve an objective diagnosis of BLI in colorectal cancer. The varying effects of diagnostic criterion on the pathologists from Japan, the USA/Canada and Europe might reflect varied interpretations of the criterion. Internationally accepted criterion should be developed by participants from around the world.

Expression of the hyaluronan-mediated motility receptor RHAMM in tumor budding cells identifies aggressive colorectal cancers.

Expression of the hyaluronan-mediated motility receptor (RHAMM, CD168) predicts adverse clinicopathological features and decreased survival for colorectal cancer (CRC) patients. Using full tissue sections, we investigated the expression of RHAMM in tumor budding cells of 103 primary CRCs to characterize the biological processes driving single-cell invasion and early metastatic dissemination. RHAMM expression in tumor buds was analyzed with clinicopathological data, molecular features and survival. Tumor budding cells at the invasive front of CRC expressed RHAMM in 68% of cases. Detection of RHAMM-positive tumor budding cells was significantly associated with poor survival outcome (P = .0312), independent of TNM stage and adjuvant therapy in multivariate analysis (P = .0201). RHAMM-positive tumor buds were associated with frequent lymphatic invasion (P = .0007), higher tumor grade (P = .0296), and nodal metastasis (P = .0364). Importantly, the prognostic impact of RHAMM expression in tumor buds was maintained independently of the number of tumor buds found in an individual case (P = .0246). No impact of KRAS/BRAF mutation, mismatch repair deficiency and CpG island methylation was observed. RHAMM expression identifies an aggressive subpopulation of tumor budding cells and is an independent adverse prognostic factor for CRC patients. These data support ongoing efforts to develop RHAMM as a target for precision therapy.

Loss of tapasin correlates with diminished CD8(+) T-cell immunity and prognosis in colorectal cancer.

BACKGROUND Tapasin is a crucial component of the major histocompatibility (MHC) class I antigen presentation pathway. Defects in this pathway can lead to tumor immune evasion. The aim of this study was to test whether tapasin expression correlates with CD8(+) cytotoxic T lymphocyte (CTL) infiltration of colorectal cancer (CRC) and overall survival. METHODS A next-generation tissue microarray (ngTMA) of 198 CRC patients with full clinicopathological information was included in this study. TMA slides were immunostained for tapasin, MHC I and CD8. Marker expression was analyzed with immune-cell infiltration, patient survival and TNM-staging. RESULTS A reduction of tapasin expression strongly correlated with venous invasion (AUC 0.682, OR 2.7, p = 0.002; 95% CI 1.7-5.0), lymphatic invasion (AUC 0.620, OR 2.0, p = 0.005; 95 % CI 1.3-3.3), distant metastasis (AUC 0.727, OR 2.9, p = 0.004; 95% CI 1.4-5.9) and an infiltrative tumor border configuration (AUC 0.621, OR 2.2, p = 0.017; 95% CI 1.2-4.4). Further, tapasin expression was associated with CD8(+) CTL infiltration (AUC 0.729, OR 5.4, p < 0.001; 95% CI 2.6-11), and favorable overall survival (p = 0.004, HR 0.6, 95% CI 0.42-0.85). CONCLUSIONS Consistent with published functional data showing that tapasin promotes antigen presentation, as well as tumor immune recognition and destruction by CD8(+) CTLs, a reduction in tapasin expression is associated with tumor progression in CRC.

FOXC2 and fluid shear stress stabilize postnatal lymphatic vasculature

Biomechanical forces, such as fluid shear stress, govern multiple aspects of endothelial cell biology. In blood vessels, disturbed flow is associated with vascular diseases, such as atherosclerosis, and promotes endothelial cell proliferation and apoptosis. Here, we identified an important role for disturbed flow in lymphatic vessels, in which it cooperates with the transcription factor FOXC2 to ensure lifelong stability of the lymphatic vasculature. In cultured lymphatic endothelial cells, FOXC2 inactivation conferred abnormal shear stress sensing, promoting junction disassembly and entry into the cell cycle. Loss of FOXC2-dependent quiescence was mediated by the Hippo pathway transcriptional coactivator TAZ and, ultimately, led to cell death. In murine models, inducible deletion of Foxc2 within the lymphatic vasculature led to cell-cell junction defects, regression of valves, and focal vascular lumen collapse, which triggered generalized lymphatic vascular dysfunction and lethality. Together, our work describes a fundamental mechanism by which FOXC2 and oscillatory shear stress maintain lymphatic endothelial cell quiescence through intercellular junction and cytoskeleton stabilization and provides an essential link between biomechanical forces and endothelial cell identity that is necessary for postnatal vessel homeostasis. As FOXC2 is mutated in lymphedema-distichiasis syndrome, our data also underscore the role of impaired mechanotransduction in the pathology of this hereditary human disease.

ICAM1 depletion reduces spinal metastasis formation in vivo and improves neurological outcome

INTRODUCTION Clinical treatment of spinal metastasis is gaining in complexity while the underlying biology remains unknown. Insufficient biological understanding is due to a lack of suitable experimental animal models. Intercellular adhesion molecule-1 (ICAM1) has been implicated in metastasis formation. Its role in spinal metastasis remains unclear. It was the aim to generate a reliable spinal metastasis model in mice and to investigate metastasis formation under ICAM1 depletion. MATERIAL AND METHODS B16 melanoma cells were infected with a lentivirus containing firefly luciferase (B16-luc). Stable cell clones (B16-luc) were injected retrogradely into the distal aortic arch. Spinal metastasis formation was monitored using in vivo bioluminescence imaging/MRI. Neurological deficits were monitored daily. In vivo selected, metastasized tumor cells were isolated (mB16-luc) and reinjected intraarterially. mB16-luc cells were injected intraarterially in ICAM1 KO mice. Metastasis distribution was analyzed using organ-specific fluorescence analysis. RESULTS Intraarterial injection of B16-luc and metastatic mB16-luc reliably induced spinal metastasis formation with neurological deficits (B16-luc:26.5, mB16-luc:21 days, p<0.05). In vivo selection increased the metastatic aggressiveness and led to a bone specific homing phenotype. Thus, mB16-luc cells demonstrated higher number (B16-luc: 1.2±0.447, mB16-luc:3.2±1.643) and increased total metastasis volume (B16-luc:2.87±2.453 mm3, mB16-luc:11.19±3.898 mm3, p<0.05) in the spine. ICAM1 depletion leads to a significantly reduced number of spinal metastasis (mB16-luc:1.2±0.84) with improved neurological outcome (29 days). General metastatic burden was significantly reduced under ICAM1 depletion (control: 3.47×10(7)±1.66×10(7); ICAM-1-/-: 5.20×10(4)±4.44×10(4), p<0.05 vs. control) CONCLUSION Applying a reliable animal model for spinal metastasis, ICAM1 depletion reduces spinal metastasis formation due to an organ-unspecific reduction of metastasis development.

Attenuated portal hypertension in germ-free mice: Function of bacterial flora on the development of mesenteric lymphatic and blood vessels

Intestinal bacterial flora may induce splanchnic hemodynamic and histological alterations that are associated with portal hypertension (PH). We hypothesized that experimental PH would be attenuated in the complete absence of intestinal bacteria. We induced prehepatic PH by partial portal vein ligation (PPVL) in germ-free (GF) or mice colonized with altered Schaedler's flora (ASF). After 2 or 7 days, we performed hemodynamic measurements, including portal pressure (PP) and portosystemic shunts (PSS), and collected tissues for histomorphology, microbiology, and gene expression studies. Mice colonized with intestinal microbiota presented significantly higher PP levels after PPVL, compared to GF, mice. Presence of bacterial flora was also associated with significantly increased PSS and spleen weight. However, there were no hemodynamic differences between sham-operated mice in the presence or absence of intestinal flora. Bacterial translocation to the spleen was demonstrated 2 days, but not 7 days, after PPVL. Intestinal lymphatic and blood vessels were more abundant in colonized and in portal hypertensive mice, as compared to GF and sham-operated mice. Expression of the intestinal antimicrobial peptide, angiogenin-4, was suppressed in GF mice, but increased significantly after PPVL, whereas other angiogenic factors remained unchanged. Moreover, colonization of GF mice with ASF 2 days after PPVL led to a significant increase in intestinal blood vessels, compared to controls. The relative increase in PP after PPVL in ASF and specific pathogen-free mice was not significantly different. CONCLUSION In the complete absence of gut microbial flora PP is normal, but experimental PH is significantly attenuated. Intestinal mucosal lymphatic and blood vessels induced by bacterial colonization may contribute to development of PH.

Indocyanine Green Fluorescence Imaging in the Surgical Management of an Iatrogenic Lymphatic Fistula: Description of a Surgical Technique

We present a case of laparoscopic surgical management of an iatrogenic lymphorrhea using indocyanine green (ICG). A case of a patient who developed recurrent symptomatic lymphorrhea after laparoscopic radical hysterectomy and bilateral pelvic lymphadenectomy for an early stage cervical cancer is presented. Intraoperative bipedal interdigital subcutaneous injection of ICG exactly localized the disrupted lymphatic duct on fluorescence imaging performed with a near-infrared laparoscopic fluorescent optic device, thus allowing a successful surgical repair.

Receptor tyrosine kinase HER2-mediated signaling pathways in metastasis and angiogenesis

Metastasis, the major cause of morbidity and mortality in most cancers, is a highly organized and organ-selective process. The receptor tyrosine kinase HER2 enhances tumor metastasis, however, its role in homing to metastatic organs is poorly understood. The chemokine receptor CXCR4 has recently been shown to mediate the malignant cancer cells to specific organs. Here we show that HER2 enhances the expression of CXCR4 by increasing CXCR4 protein synthesis and inhibiting its degradation. We also observed significant correlation between HER2 and CXCR4 expression in human breast tumor tissues, and an association between CXCR4 expression and a poor overall survival rate in patients with breast cancer. Furthermore, we found that CXCR4 is required for HER2-induced invasion, migration, and adhesion activities in vitro . Finally we established stable transfectants using retroviral RNA interference to inhibit CXCR4 expression and showed that the CXCR4 is required for HER2-mediated lung metastasis in vivo. These results provide a plausible mechanism for HER2-mediated breast tumor metastasis and homing to metastatic organs, and establish a functional link between the receptor tyrosine kinase HER2 and the chemokine receptor CXCR4 signaling pathways. ^ The HER2 overexpression activates PI-3K/Akt pathways and plays an important role in mediating cell survival and tumor development. Hypoxia inducible factors (HIF) are the key regulator for angiogenesis and energy metabolism, and thereby enhance tumor growth and metastasis. HIF activation occurs in the majority of human cancers, including the HER2 overexpressing cancer cells. Previous reports suggested that increased PI-3K/Akt may activate HIF pathway in various tumors, but the detail mechanism is still not completely understood. Here we found that HER2/PI-3K/Akt pathway induces HIF-1α activation, which is independent of hypoxia, but relatively weaker than hypoxic stimulation. This phenomenon was further observed in Akt knock out mouse embryonic fibroblast cells. The PI-3K/Akt pathway does not affect HIF-1α binding with its E3 ligase VHL, but enhances the binding affinity between HIF-1α and β unit. Furthermore, we found Akt phosphorylates HIF-1β at serine 271 and further regulated HIF transcriptional activity. Our findings provided one mechanism that HER2 induce HIF activation via Akt to promote angiogenesis, and this process is independent on hypoxia, which may have implications in the oncogenic activity of HER2 and PI-3K/Akt pathway. ^

HRGbeta1 inhibits metastasis of breast cancer cell lines, one of which exhibits lineage infidelity

Recent publications have questioned the origin of the MDA-MB-435 breast cancer cell line and have suggested that it is of melanocyte origin rather than breast epithelial origin. The data presented herein show unequivocally that MDA-MB-435 does express breast epithelial markers and produces milk-specific lipids. The data also indicated that MDA-MB-435 does express some melanocyte proteins but this expression occurs in the same MDA-MB-435 cells that express breast epithelial proteins. Although MDA-MB-435 does not strictly adhere to a breast lineage, it does retain breast specific markers and is thus valid as an experimental cell line in breast cancer studies. ^ Heregulinβ1 (HRGβ1) has been shown to both stimulate and inhibit breast tumorigenic and metasastasic phenotypes. Some studies used only the EGF-like domain of the extracellular domain of HRGβ1 while others used bacterially-expressed HRGβ1. Our in vitro data demonstrated that the full-length extracellular domain of human HRGβ1 reduced clonal growth of MDA-MB-435 breast cancer cells but stimulated apoptosis in MDA-MB-435 and MCF-7 breast cancer cells. In addition, mammalian-expressed HRGβ1 did not dramatically affect matrix metalloproteinase-9 activity but did inhibit cell motility of MDA-MB-435 and MCF-7 cells. Taken together, the in vitro data indicated that HRGβ1 inhibits metastasis-associated properties. ^ The in vivo data demonstrated that inducible expression of the full-length extracellular domain of human HRGβ1 in MDA-MB-435 cells reduced tumor volume and cell proliferation but increased apoptosis of cells injected at the mammary fat pad in nude mice. More importantly, HRGβ1 reduced the number of metastases observed by a spontaneous metastasis assay. Taken together, these data indicate that the full-length extracellular domain of human HRGβ1 has the net effect of inhibiting breast cancer metastasis. ^

High-resolution microarray analysis of chromosome 20q in human colon cancer metastasis model systems

Amplification of human chromosome 20q DNA is the most frequently occurring chromosomal abnormality detected in sporadic colorectal carcinomas and shows significant correlation with liver metastases. Through comprehensive high-resolution microarray comparative genomic hybridization and microarray gene expression profiling, we have characterized chromosome 20q amplicon genes associated with human colorectal cancer metastasis in two in vitro metastasis model systems. The results revealed increasing complexity of the 20q genomic profile from the primary tumor-derived cell lines to the lymph node and liver metastasis derived cell lines. Expression analysis of chromosome 20q revealed a subset of over expressed genes residing within the regions of genomic copy number gain in all the tumor cell lines, suggesting these are Chromosome 20q copy number responsive genes. Bases on their preferential expression levels in the model system cell lines and known biological function, four of the over expressed genes mapping to the common intervals of genomic copy gain were considered the most promising candidate colorectal metastasis-associated genes. Validation of genomic copy number and expression array data was carried out on these genes, with one gene, DNMT3B, standing out as expressed at a relatively higher levels in the metastasis-derived cell lines compared with their primary-derived counterparts in both the models systems analyzed. The data provide evidence for the role of chromosome 20q genes with low copy gain and elevated expression in the clonal evolution of metastatic cells and suggests that such genes may serve as early biomarkers of metastatic potential. The data also support the utility of the combined microarray comparative genomic hybridization and expression array analysis for identifying copy number responsive genes in areas of low DNA copy gain in cancer cells. ^

The role and mechanism of supressor of cytokine signaling 1 in brain metastasis

Brain metastasis, which occurs in 40%-60% of patients with advanced melanoma, has led directly to death in the majority of cases. Unfortunately, little is known about the biological and molecular basis of melanoma brain metastases. In our previous study, we developed a model to study human melanoma brain metastasis and found that Stat3 activity was increased in human brain metastatic melanoma cells when compared with that in cutaneous melanoma cells. The increased activation of Stat3 is also responsible for affecting melanoma angiogenesis in vivo and melanoma cell invasion in vitro and significantly affecting the expression of bFGF, VEGF, and MMP-2 in vivo and in vitro. Interestingly, a member of a new family of cytokine-inducible inhibitors of signal transduction, termed suppressors of cytokine signaling 1 (SOCS1) was found to negatively regulate the Janus kinase signal transducer and activator of transcription (Jak/STAT) signaling cascade. Here we report that restoration of SOCS1 expression by transfecting of SOCS1-expressing vector effectively inhibited melanoma brain metastasis through inhibiting Stat3 activation and further affecting melanoma angiogenesis and melanoma cell invasion in vitro, and significantly affected the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2) in vitro and in vivo. In addition, we used cDNA array to compare mRNA expression in the SOCS1-transfected and vector-transfected cell lines and found some genes are tightly correlated to the restoration of SOCS1. One of them is Caveolin-1 (Cav-1). Cav-1 was reported to function as a tumor suppressor gene by several groups. Finally, the Cav-1 expression is up-regulated in SOCS1-overexpressing cell line. Further study found the regulation of Cav-1 by SOCS1 occurs through inhibiting Stat3 activation. Activated Stat3 binds directly to Cav-1 promoter and the Cav-1 promoter within -575bp is essential for active Stat3 binding. My studies reveal that Stat3 activation and SOCS1 expression play important roles in melanoma metastases. Moreover, the expression between SOCS1, Stat3 and Cav-1 forms a feedback regulation loop. ^