Elevated Expression Of IL-8 Research Articles

Abstract PD6-05: Characterization of the immune landscape of primary vs metastatic breast cancer in a real-world data cohort

Abstract Breast cancer is considered to be a relatively immunologically “silent” tumor type. However, there is substantial heterogeneity in breast cancer, including differences in the immune microenvironment of tumors. For example, differences in the immune profile of primary and metastatic breast tumors have been observed. A deeper investigation of the immune profile of primary and metastatic breast tumors would enhance our understanding of late-stage breast cancer and provide insights that can guide the development of immunotherapies for primary or metastatic disease. In the current study, we explored differences in the immune microenvironment of primary and metastatic breast tumors in a clinico-genomic, real-world cohort (Tempus) of 420 subjects with breast cancer. Use of a real-world cohort for this study provided a diverse set of samples across subtypes, stages, and sites, and in particular a relatively large number of biopsies from metastatic sites as compared to other large breast cancer cohorts. Analyses were conducted using gene expression data generated by bulk RNA-sequencing of biopsies from lesions at primary (stage I-IV, N = 110) and metastatic (N = 310) sites, along with subject medical histories compiled from a variety of sources, including clinical records, pathology images, and lab reports. Differences in the immune microenvironment of assayed tumors were characterized by evaluating gene expression signatures representing immune-related processes as well as individual immune cell types, with emphasis on signatures known to be associated with response to checkpoint blockade, as well as differential expression analysis of individual genes. We observed higher expression of inflammation signatures in early stage and locally advanced primary tumors as compared to metastatic tumors (excluding lymph node biopsies), as well as enrichment of a number of immune cell types, including CD8+ T cells, regulatory T cells, B cells, and dendritic cells. We further examined individual metastatic sites (including lymph nodes, liver, brain, bone, and lung, among others), and noted variability in the immune contexture of metastatic tumors across sites. For example, we observed a trend to reduced inflammation and infiltration of certain cell types in brain and liver metastases as compared to primary tumors. Differential gene expression analysis of immune checkpoint molecules and other key immune-related genes was also conducted, revealing, for example, higher expression of IL-8 at certain metastatic sites; elevated IL-8 expression has been linked to reduced response to PD-1/PD-L1 agents and an immunosuppressive microenvironment. Finally, transcriptome-wide differential gene expression and gene set enrichment analyses were conducted using several gene-set and pathway databases (Gene Ontology, KEGG, Reactome, and Molecular Signatures Database hallmark collection). The results from these hypothesis-free analyses confirmed observations from the gene signature analyses described above; for example, inflammation-related pathways like the Hallmark “Interferon Gamma Response” and Gene Ontology “Regulation of Inflammatory Response” were enriched in primary tumors. Overall, the results of this study provide insights into the immune microenvironment of breast tumors and may be further used to inform the development of immune-oncology therapies in the space. Citation Format: Aparna Chhibber, Kim Van Naarden Braun, Celine Han, Han Chang, Mustimbo Roberts, Bin Li. Characterization of the immune landscape of primary vs metastatic breast cancer in a real-world data cohort [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD6-05.

Transcriptomic Analysis of Mycobacterium leprae-Stimulated Response in Peripheral Blood Mononuclear Cells Reveal Potential Biomarkers for Early Diagnosis of Leprosy.

We aimed to identify an unique host transcriptional signature in peripheral blood mononuclear cells (PBMCs) in response to Mycobacterium leprae antigens to distinguish between patients with leprosy and non-leprosy controls for early diagnosis of the disease. Sixteen individuals were enrolled in the discovery cohort [eight patients with leprosy, comprising four multibacillary (MB) and four paucibacillary (PB); and eight non-leprosy controls, comprising four healthy house contacts (HHCs) and four endemic controls (ECs)]. The differences in the transcriptome response of PBMCs to M. leprae sonicate antigen were evaluated between leprosy patients and non-leprosy controls, and 12 differentially expressed genes (CCL2/MCP-1, IL-8, JAKM, ATP, ND1, SERP, FLJ10489, LINC00659, LOC34487, LOC101928143, MIR22, and NCF1C) were identified. The accuracy of the 12 differentially expressed genes was further validated for the diagnosis of leprosy using real-time quantitative PCR in 82 individuals (13 MB, 10 PB, 37 HHCs, and 22 ECs) in the validation cohort. We found that a 5 gene signature set IL-8, CCL2/MCP-1, SERP, LINC00659 and FLJ10489 had a suitable performance in discriminating leprosy from ECs. In addition, elevated expression of IL-8, CCL2/MCP-1, SERP and LINC00659 was associated with MB diagnosis compared with ECs, whereas increased expression of IL-8, CCL2/MCP-1, SERP and FLJ10489 was found to be useful biomarkers for PB diagnosis from ECs. Moreover, we found decreased expression of NCF1C among leprosy patients could distinguish leprosy from HHCs, whereas higher expression of CCL2 among MB than PB could distinguish different leprosy patients. In conclusion, among the 12 candidate host genes identified, a three gene signature IL-8, CCL2/MCP-1, and SERP showed the best performance in distinguishing leprosy patients from healthy controls. These findings may have implications for developing a rapid blood-based test for early diagnosis of leprosy.

Activation of GSK3 Prevents Termination of TNF-Induced Signaling.

BackgroundTermination of TNF-induced signaling plays a key role in the resolution of inflammation with dysregulations leading to severe pathophysiological conditions (sepsis, chronic inflammatory disease, cancer). Since a recent phospho-proteome analysis in human monocytes suggested GSK3 as a relevant kinase during signal termination, we aimed at further elucidating its role in this context.Materials and MethodsFor the analyses, THP-1 monocytic cells and primary human monocytes were used. Staurosporine (Stauro) was applied to activate GSK3 by inhibiting kinases that mediate inhibitory GSK3α/β-Ser21/9 phosphorylation (eg, PKC). For GSK3 inhibition, Kenpaulone (Ken) was used. GSK3- and PKC-siRNAs were applied for knockdown experiments. Protein expression and phosphorylation were assessed by Western blot or ELISA and mRNA expression by qPCR. NF-κB activation was addressed using reporter gene assays.ResultsConstitutive GSK3β and PKCβ expression and GSK3α/β-Ser21/9 and PKCα/βII-Thr638/641 phosphorylation were not altered during TNF long-term incubation. Stauro-induced GSK3 activation (demonstrated by Bcl3 reduction) prevented termination of TNF-induced signaling as reflected by strongly elevated IL-8 expression (used as an indicator) following TNF long-term incubation. A similar increase was observed in TNF short-term-exposed cells, and this effect was inhibited by Ken. PKCα/β-knockdown modestly increased, whereas GSK3α/β-knockdown inhibited TNF-induced IL-8 expression. TNF-dependent activation of two NF-κB-dependent indicator plasmids was enhanced by Stauro, demonstrating transcriptional effects. A TNF-induced increase in p65-Ser536 phosphorylation was further enhanced by Stauro, whereas IκBα proteolysis and IKKα/β-Ser176/180 phosphorylation were not affected. Moreover, PKCβ-knockdown reduced levels of Bcl3. A20 and IκBα mRNA, both coding for signaling inhibitors, were dramatically less affected under our conditions when compared to IL-8, suggesting differential transcriptional effects.ConclusionOur results suggest that GSK3 activation is involved in preventing the termination of TNF-induced signaling. Our data demonstrate that activation of GSK3 – either pathophysiologically or pharmacologically induced – may destroy the finely balanced condition necessary for the termination of inflammation-associated signaling.

Self-seeding circulating tumor cells promote the proliferation and metastasis of human osteosarcoma by upregulating interleukin-8

Most circulating tumor cells (CTCs) die during the process of metastasis, but self-seeding CTCs can invade the primary tumor or form clinically meaningful metastases. This study aimed to evaluate the capacity of self-seeding CTCs to promote osteosarcoma growth and lung metastasis and to clarify the specific role of interleukin (IL)-8 in CTC self-seeding. We successfully isolated and cultured self-seeding CTCs through a self-seeding nude mouse model established using green fluorescent protein (GFP)-labeled F5M2 cells and found that self-seeding CTCs exhibit increased cellular proliferation, migration, and invasion in vitro, increased tumor growth and lung metastasis in mice, and increased IL-8 expression. Furthermore, suppressing IL-8 inhibited tumor growth and metastasis and reduced CTC seeding in primary tumors in vitro and in vivo. In osteosarcoma patients, IL-8 levels significantly correlated with the Enneking stage and metastasis. These findings demonstrate that self-seeding osteosarcoma CTCs can promote tumor growth and lung metastasis through IL-8. Their increased metastatic potential and elevated IL-8 expression suggest a novel strategy for future therapeutic interventions to prevent osteosarcoma progression and metastasis.

Effects of Live and Heat-Inactivated E. coli Strains and Their Supernatants on Immune Regulation in HT-29 Cells.

Probiotics are considered to have a beneficial impact on humans, but in some cases, administration of live microorganisms might be risky. In the present study, immunomodulatory effects of different Escherichia coli strains and their super-natants were examined under different inflammatory conditions with living and heat-inactivated strains. HT-29 cells were incubated with E. coli strains (S2-G1, S2-G3, S2-G4 and S2-G8) and their supernatants with or without stimulation with tumor necrosis factor alpha (TNF-α) or interleukin (IL)-1β. Quantification of IL-8 secretion and gene expression was performed by enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR). IL-8 secretion by TNF-α- and IL-1β-stimulated cells was attenuated by all four live strains. In contrast, heat inactivation resulted in an elevated IL-8 expression and secretion in unstimulated cells and did not maintain the anti-inflammatory effect of live bacteria in cytokine-stimulated cells. The supernatant of the live S2-G3 led to an elevated IL-8 secretion in unstimulated and IL-1β-stimulated cells but not in TNF-α-stimulated cells. Live bacteria of all strains might induce an immunosuppressive effect after stimulation of HT-29 cells, whereas heat inactivation and the supernatant seem to induce an elevated immune response. These findings might have an impact depending on the indication and purpose of administration.

Taxotere-induced elevated expression of IL8 in carcinoma-associated fibroblasts of breast invasive ductal cancer.

Breast cancer is the most common malignant tumor in women worldwide, and accounts for an estimated 29% of new cases and 15% of cancer-associated mortalities each year. Invasive ductal carcinoma represents 70-80% of all breast cancer cases, which are responsible for the majority of breast cancer fatalities. Though great progress has been made in understanding the tumorigenesis and development of breast cancer, problems surrounding treatment persist. It was previously reported that carcinoma-associated fibroblasts (CAFs) may be closely associated with chemotherapy resistance. In the present study, primary-cultured CAFs from surgically resected breast invasive ductal cancer tissues were prepared and tested to clarify the change of gene expression profile following treatment with 20 ng/ml Taxotere® for 24 h through microarray analysis. In addition, quantitative polymerase chain reaction and western blotting were performed to compare the gene and protein expression of the candidate gene in CAFs prior to and following Taxotere treatment. Based on the obtained data, 35 differentially expressed genes were identified, including ACTA2, ACTC1, ACTG, ALDH1B1, AMY1A, C5orf13, CNN1, CXCR7, DDAH1, FGF1, PDLIM3, MAMLD1, MYH11, OXTR, PDLIM5, RARRES1, SERPINA3, TRIL, C14orf43, C1orf51, CXCL12, CXCL2, EGR2, EGR3, IER3, interleukin (IL)8, IRF1, JUNB, MMP1, NAV2, NFKBIA, NFKBIZ, TRIB1, WNT16 and ZC3H12A. It was observed that the expression of the candidate gene IL8 in the CAFs of breast invasive cancer following treatment with Taxotere was increased (P<0.05). Overall, elevated expression of IL8 induced by Taxotere in CAFs potentially supports the association between IL8 and chemotherapy response.

M3 mAChR-mediated IL-8 expression through PKC/NF-κB signaling pathways

M3 muscarinic acetylcholine receptor (mAChR) plays an important role in the regulation of cytokine production in inflammatory diseases. In this study, we explored the precise role of M3 mAChR under stimulation with agonist in IL-8 expression and of the signaling pathway involved in this process. Recombinant U2OS cells stably expressing M3 mAChR as a model system were stimulated by carbachol to evaluate the role of M3 mAChR in the expression of IL-8. Activation of M3 mAChR with carbachol increased both IL-8 mRNA and protein expression in a concentration-dependent manner. Elevated IL-8 expression was completely antagonized by atropine, 4-DAMP and tiotropium. M3 mAChR-mediated IL-8 expression was almost completely inhibited by the NF-κB inhibitor BAY11-7082 and, to a lesser extent, by U0126, SB203580, and SP600125, which are inhibitors for ERK1/2, p38, and JNK, respectively. Furthermore, M3 mAChR-mediated NF-κB activation and IL-8 expression were simultaneously attenuated by the PKC inhibitor calphostin C, whereas PMA, a PKC activator, mimicked the effects of carbachol, inducing IL-8 expression. Our findings offer insights into the specific and critical role of M3 mAChR in regulating inflammatory response and indicate M3 mAChR/PKC/NF-κB signaling axis driven by endogenous acetylcholine as a potential therapeutic targets for inflammatory diseases.

Seminal plasma induces angiogenic chemokine expression in cervical cancer cells and regulates vascular function

Cervical cancer is one of the leading gynecological malignancies in women. We have recently shown that seminal plasma (SP) can regulate the inflammatory cyclooxygenase-prostaglandin pathway and enhance the growth of cervical epithelial tumours in vivo by promoting cellular proliferation and alteration of vascular function. This study investigated the molecular mechanism whereby SP regulates vascular function using an in vitro model system of HeLa cervical adenocarcinoma cells and human umbilical vein endothelial cells (HUVECs). We found that SP rapidly enhanced the expression of the angiogenic chemokines, interleukin (IL)-8 and growth regulated oncogene alpha (GRO) in HeLa cells in a time-dependent manner. We investigated the molecular mechanism of SP-mediated regulation of IL-8 and GRO using a panel of chemical inhibitors of cell signalling. We found that treatment of HeLa cells with SP elevated expression of IL-8 and GRO by transactivation of the epidermal growth factor receptor, activation of extracellular signal-regulated kinase and induction of cyclooxygenase enzymes and nuclear factor kappa B. We investigated the impact of IL-8 and GRO, released from HeLa cells after treatment with SP, on vascular function using a co-culture model system of conditioned medium (CM) from HeLa cells, treated with or without SP, and HUVECs. We found that CM from HeLa cells induced the arrangement of endothelial cells into a network of tube-like structures via the CXCR2 receptor on HUVECs. Taken together our data outline a molecular mechanism whereby SP can alter vascular function in cervical cancers via the pro-angiogenic chemokines, IL-8 and GRO.

Nickel promotes the invasive potential of human lung cancer cells via TLR4/MyD88 signaling

Epidemiological studies have demonstrated a close correlation between nickel exposure and the incidence of lung cancer. Several studies have suggested that nickel contributes to tumor progression of human lung cancer. In this in vitro study, we found that nickel, as nickel chloride, could significantly enhance the invasive potential of human lung cancer cells, accompanied by elevated expression of IL-8, TGF-β, MMP2 and MMP9 in human lung cancer cells. Importantly, we demonstrated that nickel could activate TLR4 signaling in human lung cancer cells. Further studies showed that the TLR4/MyD88 signaling conferred the enhanced invasive potential of human lung cancer cells induced by nickel. Finally, we revealed that the p38MAPK pathway and NF-kB pathway were necessary for the enhanced invasive potential of human lung cancer cells induced by nickel. Our data provide a mechanistic explanation for nickel induced invasion of human lung cancer, and they suggest new strategies for nickel-related lung cancer clinical biotherapies.

Loss of CFTR results in reduction of histone deacetylase 2 in airway epithelial cells

Inflammatory cytokines, particularly the neutrophil chemoattractant IL-8, are elevated in the cystic fibrosis (CF) airway, even in the absence of detectable infection. The transcriptional regulation of many inflammatory genes, including IL8 (CXCL8), involves chromatin remodeling through histone acetylation. NF-kappaB is known to facilitate histone acetylation of IL8 and other proinflammatory gene promoters, but we find that increased NF-kappaB activation cannot explain the elevated IL8 expression and promoter acetylation seen in CFTR-deficient cells. Recognized components of the NF-kappaB-coactivator complex, acetyltransferase CBP, p300, and the histone deacetylase HDAC1, are unchanged by CFTR activity. However, we find that the histone acetyltransferase (HAT)/HDAC balance is sensitive to CFTR function, as cells with reduced or absent CFTR function have decreased HDAC2 protein, resulting in hyperacetylation of the IL8 promoter and increased IL8 transcription. Reduced HDAC2 and HDAC2 activity, but not HDAC2 mRNA, is observed in cells deficient in CFTR. Suppressing HDAC2 expression with HDAC2 short hairpin RNA (shRNA) results in increased IL8 expression and promoter acetylation comparable with CFTR-deficient cells. Treating CFTR-deficient cells with N-acetyl-cysteine (NAC) increases HDAC2 expression to near control levels. Our data suggest that there is an intrinsic alteration in the HAT/HDAC balance in cells lacking CFTR function in vitro and in native CF tissue and that oxidative stress is likely contributing to this alteration. This mechanism, found in other inflammatory airway diseases, provides an explanation for the apparent dysregulation of inflammatory mediators seen in the CF airway, as reduced histone deacetylation would potentially influence many genes.

620 Elevated expression of IL-8 and IL-8 receptors in prostate cancer cells correlates with disease progression and resistance to oxaliplatin

620 Elevated expression of IL-8 and IL-8 receptors in prostate cancer cells correlates with disease progression and resistance to oxaliplatin

Identification of interleukin-8 as estrogen receptor-regulated factor involved in breast cancer invasion and angiogenesis by protein arrays.

With the goal of identifying key factors involved in human breast cancer progression, we applied human cytokine antibody arrays we have developed to screen cytokine expression levels in human breast cancer cell lines and identified interleukin (IL)-8 as a key factor involved in breast cancer invasion and angiogenesis. Elevated expression of IL-8 in breast cancer cells was associated with breast cancer invasiveness and angiogenesis. Neutralization of antibody against IL-8 specifically blocked IL-8-mediated tumor cell invasion and angiogenesis. Furthermore, IL-8 levels in human breast cancer cells were closely related to estrogen receptor (ER) status. ER positive cells expressed low levels of IL-8 whereas ER negative cells expressed high levels of IL-8. Expression of exogenous ERalpha substantially inhibited IL-8 expression. Our findings raise intriguing questions regarding the role of IL-8 in the development and progression of human breast cancer in association with ER status.