Autocrine regulation of interleukin-8 production in human monocytes

Interleukin (IL)-8 is a C-X-C chemokine that plays an important role in acute inflammation through its G protein-coupled receptors CXCR1 and CXCR2. In this study, we investigated the role of IL-8 as an autocrine regulator of IL-8 production and the signaling mechanisms involved in human peripheral blood mononuclear cells (MNCs). Sepharose-immobilized IL-8 stimulated a sevenfold increase in IL-8 production within 2 h. IL-8 induced the expression of its own message, and IL-8 biosynthesis was inhibited by cycloheximide and actinomycin D, indicating de novo RNA and protein synthesis. In contrast to MNCs, polymorphonuclear neutrophils did not respond to the immobilized IL-8 with IL-8 production despite cell surface expression of CXCR1 and CXCR2. Melanoma growth-stimulatory activity/growth-related protein-alpha (MGSA/GROalpha), which binds CXCR2 but not CXCR1, was unable to either stimulate IL-8 secretion in MNCs or desensitize these cells to respond to immobilized IL-8. The involvement of mitogen-activated protein kinase (MAPK) in IL-8-induced IL-8 biosynthesis was suggested by the ability of PD-98059, an inhibitor of MAPK kinase, to block this function. Furthermore, IL-8 induced a significant increase in extracellular signal-regulated kinase 2 phosphorylation, whereas MGSA/GROalpha was much less effective. These findings support the role of IL-8 as an autocrine regulator of IL-8 production and suggest that this function is mediated by CXCR1 through activation of MAPK.     

Autocrine motility factor stimulates a three-fold increase in inositol trisphosphate in human melanoma cells

The biochemical pathways through which tumor cell locomotion is mediated are poorly understood. Autocrine motility factor (AMF), which is produced by and stimulates motility in A2058 human melanoma cells, was used to characterize phosphoinositide (PtdIns) metabolism activated in association with tumor cell motility. AMF stimulated up to a 400% increase in de novo incorporation of 3H-myo-inositol into cellular lipids beginning 40 minutes after exposure. In cells prelabeled with 3H-myo-inositol, AMF stimulated a 200% increase in total inositol phosphates (inositol monophosphate, InsP1; inositol bisphosphate, InsP2; inositol trisphosphate, InsP3) after 90 minutes of exposure, with a 300% maximal increase in InsP3 at 120 minutes. InsP1 and InsP2 were maximally increased 130% of control values. Treatment with AMF stimulated a parallel dose-dependent increase in both motility and PtdIns levels. We have shown previously that the A2058 motile response to AMF is inhibited markedly by cell pretreatment with pertussis toxin (PT). Inositol phosphate production was inhibited by a 2-hour pretreatment of cells with PT (0.5 microgram/ml). PT treatment of A2058 membranes was associated with ADP-ribosylation of a 40-kDa protein consistent with the presence of an alpha subunit of a guanine nucleotide-binding protein (G protein). These data indicate that AMF elicits increases in cell motility and phosphoinositide metabolism via a PT-sensitive G protein signal transduction pathway.     

IL-8 expression in malignant melanoma: implications in growth and metastasis

This review article has described briefly studies supporting the concept that IL-8 expression and its regulation by inflammatory cytokines like IL-1 may play an important role in controlling the phenotypes associated with melanoma progression and metastasis. It is clear from the experiments presented here that IL-8 is an important autocrine multifunctional cytokine that modulates melanoma/cell proliferation, migration by induction of extracellular matrix degradation enzymes and induces neovascularization, all of which are critical for melanoma growth and metastasis. In addition, their expression in melanoma tumor specimens suggests an association between IL-8 expression and tumor aggressiveness. Further, inflammatory cytokines produced by either tumor cells or stromal cells may regulate IL-8 expression, which can control melanoma growth and enhance our current knowledge regarding melanoma progression and metastasis. Understanding these events and their significance will allow us to design novel therapeutic approaches for treatment of melanoma.     

Enrichment of Foxp3+ CD4 regulatory T cells in migrated T cells to IL-6- and IL-8-expressing tumors through predominant induction of CXCR1 by IL-6

Analysis of cytokine and chemokine production by tumor cell lines including five lung cancers, a malignant mesothelioma, and a malignant melanoma recently established in our laboratory showed rather high production of IL-8 in all tumors and IL-6 in one lung cancer, the malignant mesothelioma, and the malignant melanoma. We investigated the migration of PBMCs to these tumor cells using Transwell plates and showed enrichment of Foxp3(+) CD4 regulatory T cells (Tregs) in migrated T cells to both IL-6- and IL-8-producing tumors. Marked induction of CXCR1 expression on Foxp3(+) CD4 Tregs by IL-6 followed by IL-8-mediated migration appeared to be responsible for enriched migration. Frequent production of IL-8 by the tumors and Treg migration to those tumors through induction of IL-8R expression by IL-6 is one of the mechanisms for tumor escape.     

Autocrine/paracrine actions of growth hormone in human melanoma cell lines

Melanoma is the most aggressive skin cancer. Its aggressiveness is most commonly attributed to ERK pathway mutations leading to constitutive signaling. Though initial tumor regression results from targeting this pathway, resistance often emerges. Interestingly, interrogation of the NCI-60 database indicates high growth hormone receptor (GHR) expression in melanoma cell lines. To further characterize melanoma, we tested responsiveness to human growth hormone (GH). GH treatment resulted in GHR signaling and increased invasion and migration, which was inhibited by a GHR monoclonal antibody (mAb) antagonist in WM35, SK-MEL 5, SK-MEL 28 and SK-MEL 119 cell lines. We also detected GH in the conditioned medium (CM) of human melanoma cell lines. GHR, JAK2 and STAT5 were basally phosphorylated in these cell lines, consistent with autocrine/paracrine GH production. Together, our results suggest that melanomas are enriched in GHR and produce GH that acts in an autocrine/paracrine manner. We suggest that GHR may constitute a therapeutic target in melanoma.     

自分泌运动因子在肝细胞癌侵袭及转移中的作用

目的:探讨自分泌运动因子(AMF)在人肝细胞癌侵袭和转移中的作用。方法:人肝细胞系LO2和人肝细胞癌细胞株MHCC97-H作为实验材料,检测二者AMF的表达水平;设计并合成针对AMF基因序列的双链小干扰RNA转染高转移性人肝癌细胞株MHCC97-H,Western blot检测AMF基因的蛋白的表达水平;通过MTT实验检测转染后细胞的增殖力;通过体外Transwell小室对比沉默AMF基因前后的肝癌细胞的迁移力和侵袭力;最后用细胞悬液皮下接种小鼠,观察沉默AMF基因前后肝细胞的成瘤能力。结果:AMF在MHCC97-H的表达量较高;将双链小干扰RNA转入MHCC97-H后,AMF的表达显著降低(P0.05);沉默AMF基因序列后,MHCC97-H的增殖力、迁移力和侵袭力均有明显下降(P0.05);用细胞悬液皮下接种小鼠沉默AMF基因的MHCC97-H形成的肿瘤体积小于对照组(P0.05)。结论:AMF基因可调节肝癌细胞的迁移和侵袭。     

EMMPRIN Promotes Melanoma Cells Malignant Properties through a HIF-2alpha Mediated Up-Regulation of VEGF-Receptor-2

EMMPRIN''s expression in melanoma tissue was reported to be predictive of poor prognosis. Here we demonstrate that EMMPRIN up-regulated VEGF receptor-2 (VEGFR-2) in two different primary melanoma cell lines and consequently increased migration and proliferation of these cells while inhibiting their apoptosis. SiRNA inhibition of VEGFR-2 expression abrogated these EMMPRIN effects. EMMPRIN regulation of VEGFR-2 was mediated through the over-expression of HIF-2α and its translocation to the nucleus where it forms heterodimers with HIF-1β. These results were supported by an in vivo correlation between the expression of EMMPRIN with that of VEGFR-2 in human melanoma tissues as well as with the extent of HIF-2α localization in the nucleus. They demonstrate a novel mechanism by which EMMPRIN promotes tumor progression through HIF-2α/VEGFR-2 mediated mechanism, with an autocrine role in melanoma cell malignancy. The inhibition of EMMPRIN in cancer may thus simultaneously target both the VEGFR-2/VEGF system and the matrix degrading proteases to block tumor cell growth and invasion.     

Autocrine motility factor stimulates the invasiveness of malignant cells as well as up-regulation of matrix metalloproteinase-3 expression via a MAPK pathway

The autocrine motility factor (AMF) is a multifunctional protein that is involved in tumor progression including enhanced invasiveness via induction of matrix metalloproteinase-3 (MMP3). The increase in MMP3 was found in an AMF-high production tumor cell line, and c-Jun, c-Fos and mitogen-activated protein kinases (MAPKs) were also highly phosphorylated compared with the parent line. AMF stimulation induced the rapid phosphorylation of the cellular MAPK cascade and MMP3 secretion, which was blocked using a specific MAPK inhibitor. Results of this study suggest that AMF stimulation stimulates MMP3 expression via a MAPK signaling pathway.     

Tumor autocrine motility factor induces hyperpermeability of endothelial and mesothelial cells leading to accumulation of ascites fluid

Accumulation of ascites fluid often observed in some solid tumors is one of the most devastating conditions of a patient's difficulty in responding to treatment, and to a decrease in the quality of life. Various factors are thought to be associated with the formation of cancer-induced fluid accumulation and hyperpermeability of a blood vessel is thought to go with this process. Here, we report a new factor that is involved in this process, e.g., autocrine motility factor (AMF). AMF is a tumor-related cytokine which stimulates the tumor cell locomotion and migration and promotes tumor cell invasion during metastasis. AMF secretion and its receptor (AMFR) expression in tumor cells are closely correlated with disease aggravation of convalescence. The response of endothelial or mesothelial cellular morphological alternation to AMF leads to motile enhancement and vascular permeability. Tumor AMF induces gaps in an endothelial or mesothelial monolayer by stimulating a cellular movement, and accelerates the ascites accumulation. And treatment experiment with anti-AMF antibody succeeded in the reduction of the ascites accumulation, which renders AMF to the target molecule. It is suggested that AMF is one of the significant factors which relates to various pathological malignancies induced by tumor mass, and understanding of its function could benefit prognosis and treatment.     

Autocrine motility factor/phosphoglucose isomerase regulates ER stress and cell death through control of ER calcium release

Autocrine motility factor/ phosphoglucose isomerase (AMF/PGI) promotes cell survival by the pAkt survival pathway. Its receptor, gp78/AMFR, is an E3 ubiquitin ligase implicated in endoplasmic reticulum (ER)-associated protein degradation. We demonstrate here that AMF/PGI also protects against thapsigargin (TG)- and tunicamycin (TUN)-induced ER stress and apoptosis. AMF/PGI protection against the ER stress response is receptor mediated as it is not observed in gp78/AMFR-knockdown HEK293 cells. However, AMF/PGI protection against the ER stress response by TG and TUN was mediated only partially through PI3K/Akt activation. AMF/PGI reduction of the elevation of cytosolic calcium in response to either TG or inositol 1,4,5-trisphosphate receptor activation with ATP was gp78/AMFR-dependent, independent of mitochondrial depolarization and not associated with changes in ER calcium content. These results implicate regulation of ER calcium release in AMF/PGI protection against ER stress and apoptosis. Indeed, sequestration of cytosolic calcium with BAPTA-AM limited the ER stress response. Importantly, elevation of cytosolic calcium upon treatment with the calcium ionophore ionomycin, while not inducing an ER stress response, did prevent AMF/PGI protection against ER stress. By regulating ER calcium release, AMF/PGI interaction with gp78/AMFR therefore protects against ER stress identifying novel roles for these cancer-associated proteins in promoting tumor cell survival.     

Expression and secretion of gro/MGSA by stimulated human endothelial cells.

Melanoma growth stimulatory activity factor (MGSA) is a polypeptide which was initially isolated from Hs294 human melanoma cells. Its sequence is identical to the deduced amino acid sequence of the human gro cDNA, isolated from a human tumor cell line. MGSA stimulates the proliferation of malignant melanoma cells, but its function for normal cells has not been defined. Here we report that human umbilical vein endothelial cells are capable of synthesizing and secreting MGSA. The expression and secretion of MGSA are strongly induced by factors often involved in inflammation such as IL-1, TNF, LPS and thrombin. The induction of MGSA mRNA is dose and time dependent and is independent of new protein synthesis. This stimulation could be mimicked by TPA, suggesting that the action could be mediated through activation of protein kinase C. Furthermore, addition of MGSA to the endothelial cell cultures induces gro/MGSA gene expression, implying that an autocrine mechanism exists. Our data suggest that the protein encoded by gro/MGSA mRNA may play a role in inflammation and exert its effects on endothelial cells in an autocrine fashion.     

Autocrine and paracrine regulation by cytokines and growth factors in melanoma

Tumour development and progression involves the expression of oncogenes and inactivation of tumour suppressor genes, leading to the appearance of multiple malignant characteristics. Malignant melanoma cells express different growth factors and cytokines and their receptors in respective stages of tumour progression, which by autocrine and paracrine effects enable them to grow autonomously and confer competence to metastasis. Autocrine growth factors (bFGF, MGSA/GRO, IL-8 and sometimes IL-6, PDGF-A, IL-10) produced by melanoma cells stimulate proliferation of the producing cell itself, while paracrine growth factors (for example PDGF, EGF, TGF-beta, IL-1, GM-CSF, IGF-I, NGF, VEGF) modulate the microenvironment to the benefit of tumour growth and invasion. Paracrine effects include angiogenesis, stroma formation, modulation of host immune response, activation of proteolytic enzymes, adhesion or motility and metastasis formation. Some growth factors have inhibitory effects on melanocytes and early lesions (IL-1, IL-6, TGF-beta, OSM, TNF and IFN) but not on advanced stage melanomas, and in some cases they switch to autocrine stimulator (IL-6, TGF-beta). Understanding the involvement of different growth factors and cytokines in the molecular mechanism of melanoma progression will help to provide an insight into new future therapeutic approaches for melanoma.     

Autocrine IL-6/STAT3 signaling aids development of acquired drug resistance in Group 3 medulloblastoma

Medulloblastoma (MB) is a high-grade pediatric brain malignancy that originates from neuronal precursors located in the posterior cranial fossa. In this study, we evaluated the role of STAT3 and IL-6 in a tumor microenvironment mediated drug resistance in human MBs. We established that the Group 3 MB cell line, Med8A, is chemosensitive (hence Med8A-S), and this is correlated with a basal low phosphorylated state of STAT3, while treatment with IL-6 induced robust increases in pY705-STAT3. Via incremental selection with vincristine, we derived the stably chemoresistant variant, Med8A-R, that exhibited multi-drug resistance, enhanced IL-6 induced pY705-STAT3 levels, and increased IL6R expression. Consequently, abrogation of STAT3 or IL6R expression in Med8A-R led to restored chemosensitivity to vincristine, highlighting a prominent role for canonical IL-6/STAT3 signaling in acquired drug resistance. Furthermore, Med8A-S subjected to conditioning exposure with IL-6, termed Med8A-IL6+ cells, exhibited enhanced vincristine resistance, increased expression of pY705-STAT3 and IL6R, and increased secretion of IL-6. When cocultured with Med8A-IL6+ cells, Med8A-S cells exhibited increased pY705-STAT3 and increased IL-6 secretion, suggesting a cytokine feedback loop responsible for amplifying STAT3 activity. Similar IL-6 induced phenomena were also observed in the Group 3 MB cell lines, D283 and D341, including increased pY705-STAT3, drug resistance, IL-6 secretion and IL6R expression. Our study unveiled autocrine IL-6 as a promoter of STAT3 signaling in development of drug resistance, and suggests therapeutic benefits for targeting the IL-6/STAT3 signaling axis in Group 3 MBs.Subject terms: Cancer microenvironment, CNS cancer, Paediatric cancer     

Melanoma cell extravasation under flow conditions is modulated by leukocytes and endogenously produced interleukin 8

Attachment of tumor cells to the endothelium (EC) under flow conditions is critical for the migration of tumor cells out of the vascular system to establish metastases. Innate immune system processes can potentially promote tumor progression through inflammation dependant mechanisms. White blood cells, neutrophils (PMN) in particular, are being studied to better understand how the host immune system affects cancer cell adhesion and subsequent migration and metastasis. Melanoma cell interaction with the EC is distinct from PMN-EC adhesion in the circulation. We found PMN increased melanoma cell extravasation, which involved initial PMN tethering on the EC, subsequent PMN capture of melanoma cells and maintaining close proximity to the EC. LFA-1 (CD11a/CD18 integrin) influenced the capture phase of PMN binding to both melanoma cells and the endothelium, while Mac-1 (CD11b/CD18 integrin) affected prolonged PMN-melanoma aggregation. Blocking E-selectin or ICAM-1 (intercellular adhesion molecule) on the endothelium or ICAM-1 on the melanoma surface reduced PMN-facilitated melanoma extravasation. Results indicated a novel finding that PMN-facilitated melanoma cell arrest on the EC could be modulated by endogenously produced interleukin-8 (IL-8). Functional blocking of the IL-8 receptors (CXCR1 and CXCR2) on PMN, or neutralizing soluble IL-8 in cell suspensions, significantly decreased the level of Mac-1 up-regulation on PMN while communicating with melanoma cells and reduced melanoma extravasation. These results provide new evidence for the complex role of hemodynamic forces, secreted chemokines, and PMN-melanoma adhesion in the recruitment of metastatic cancer cells to the endothelium in the microcirculation, which are significant in fostering new approaches to cancer treatment through anti-inflammatory therapeutics.     

Proinflammatory S100 proteins regulate the accumulation of myeloid-derived suppressor cells

Chronic inflammation is a complex process that promotes carcinogenesis and tumor progression; however, the mechanisms by which specific inflammatory mediators contribute to tumor growth remain unclear. We and others recently demonstrated that the inflammatory mediators IL-1beta, IL-6, and PGE(2) induce accumulation of myeloid-derived suppressor cells (MDSC) in tumor-bearing individuals. MDSC impair tumor immunity and thereby facilitate carcinogenesis and tumor progression by inhibiting T and NK cell activation, and by polarizing immunity toward a tumor-promoting type 2 phenotype. We now show that this population of immature myeloid cells induced by a given tumor share a common phenotype regardless of their in vivo location (bone marrow, spleen, blood, or tumor site), and that Gr1(high)CD11b(high)F4/80(-)CD80(+)IL4Ralpha(+/-)Arginase(+) MDSC are induced by the proinflammatory proteins S100A8/A9. S100A8/A9 proteins bind to carboxylated N-glycans expressed on the receptor for advanced glycation end-products and other cell surface glycoprotein receptors on MDSC, signal through the NF-kappaB pathway, and promote MDSC migration. MDSC also synthesize and secrete S100A8/A9 proteins that accumulate in the serum of tumor-bearing mice, and in vivo blocking of S100A8/A9 binding to MDSC using an anti-carboxylated glycan Ab reduces MDSC levels in blood and secondary lymphoid organs in mice with metastatic disease. Therefore, the S100 family of inflammatory mediators serves as an autocrine feedback loop that sustains accumulation of MDSC. Since S100A8/A9 activation of MDSC is through the NF-kappaB signaling pathway, drugs that target this pathway may reduce MDSC levels and be useful therapeutic agents in conjunction with active immunotherapy in cancer patients.     

Induction of haptotactic migration of melanoma cells by neutrophil activating protein/interleukin-8

Natural or recombinant neutrophil activating cytokine (IL-8) induced migration across polycarbonate filters of human A 2058 melanoma cells. Anti-IL-8 antibodies blocked IL-8 induced melanoma cell migration. Checkerboard experiments revealed a gradient-dependent response of A2058 melanoma cells to IL-8. Filters exposed to IL-8 and washed supported melanoma cell migration, thus implying a haptotactic component in the response. The homologous polypeptide platelet factor 4 was inactive. The observation that IL-8 affects melanoma cells emphasizes the need for a comprehensive analysis of the spectrum of action of platelet factor 4-related peptides. The effect of the inflammatory cytokine IL-8 on melanoma cells may be relevant to augmented secondary localization of tumors at sites of inflammation.     

Autocrine transforming growth factor-beta signaling mediates Smad-independent motility in human cancer cells

Transforming growth factor-beta (TGF-beta) is a pleiotropic growth factor that plays a critical role in modulating cell growth, differentiation, and plasticity. There is increasing evidence that after cells lose their sensitivity to TGF-beta-mediated growth inhibition, autocrine TGF-beta signaling may potentially promote tumor cell motility and invasiveness. To understand the molecular mechanisms by which autocrine TGF-beta may selectively contribute to tumor cell motility, we have generated MDA-MB-231 breast cancer cells stably expressing a kinase-inactive type II TGF-beta receptor (T beta RII-K277R). Our data indicate that T beta RII-K277R is expressed, can associate with the type I TGF-beta receptor, and block both Smad-dependent and -independent signaling pathways activated by TGF-beta. In addition, wound closure and transwell migration assays indicated that the basal migratory potential of T beta RII-K277R expressing cells was impaired. The impaired motility of T beta RII-K277R cells could be restored by reconstituting TGF-beta signaling with a constitutively active TGF-beta type I receptor (ALK5(TD)) but not by reconstituting Smad signaling with Smad2/4 or Smad3/4 expression. In addition, the levels of ALK5(TD) expression sufficient to restore motility in the cells expressing T beta RII-K277R were associated with an increase in phosphorylation of Akt and extracellular signal-regulated kinase 1/2 but not Smad2. These data indicate that different signaling pathways require different thresholds of TGF-beta activation and suggest that TGF-beta promotes motility through mechanisms independent of Smad signaling, possibly involving activation of the phosphatidylinositol 3-kinase/Akt and/or mitogen-activated protein kinase pathways.     

Interleukin 6 mediates production of interleukin 10 in metastatic melanoma

We previously reported that substantial amounts of IL-10, an immunomodulatory cytokine, are produced by cell suspensions of fresh human metastatic melanoma tissues. Production diminished with continuous culturing of cells, which suggests a pivotal interactive role between melanoma cells and the tumor microenvironment. In this study, we found that the culture media obtained from LPS-stimulated peripheral blood mononuclear cells induced IL-10 production by metastatic melanoma cells. Of the multiple cytokines present in the conditioned culture media, IL-6 was identified as the inducer of IL-10 production. A neutralizing antibody against IL-6 completely blocked the conditioned medium-induced IL-10 production. Metastatic melanoma cells that constitutively produce low amount of IL-10 increased IL-10 production in response to recombinant human IL-6 in a dose-dependent fashion. The response to exogenously added IL-6 was less significant in melanoma cells that produced high amounts of IL-6, probably due to pre-existing autocrine stimulation of IL-10 by endogenous IL-6. On the other hand, metastatic melanoma cells that do not constitutively produce IL-10 protein did not respond to exogenous IL-6. In IL-6-responsive melanoma cells, IL-6 increased STAT3 phosphorylation and inhibition of STAT3 signaling using siRNA or inhibitors for JAKs diminished IL-6-induced IL-10 production. In addition, inhibition of MEK and PI3K, but not mTOR, interfered with IL-10 production. Taken together, the data suggest that blocking of these signals leading to IL-10 production is a potential strategy to enhance an anti-melanoma immune response in metastatic melanoma.