Exploring the Role of IL-32 in HIV-Related Kaposi Sarcoma
2017; Elsevier BV; Volume: 188; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2017.08.033
ISSN1525-2191
AutoresGeorge Semango, Bas Heinhuis, Theo S. Plantinga, Willeke A.M. Blokx, Gibson Kibiki, Tolbert Sonda, Daudi Mavura, Elisante J. Masenga, Mramba Nyindo, André van der Ven, Leo A. B. Joosten,
Tópico(s)Immune Cell Function and Interaction
ResumoThe intracellular proinflammatory mediator IL-32 is associated with tumor progression; however, the mechanisms remain unknown. We studied IL-32 mRNA expression as well as expression of other proinflammatory cytokines and mediators, including IL-1α, IL-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α, the proangiogenic and antiapoptotic enzyme cyclooxygenase-2, the IL-8 receptor C-X-C chemokine receptor (CXCR) 1, and the intracellular kinase focal adhesion kinase-1. The interaction of IL-32 expression with expression of IL-6, TNF-α, IL-8, and cyclooxygenase-2 was also investigated. Biopsy specimens of 11 HIV-related, 7 non–HIV-related Kaposi sarcoma (KS), and 7 normal skin tissues (NSTs) of Dutch origin were analyzed. RNA was isolated from the paraffin material, and gene expression levels of IL-32 α, β, and γ isoforms, IL1a, IL1b, IL6, IL8, TNFA, PTGS2, CXCR1, and PTK2 were determined using real-time quantitative PCR. Significantly higher expression of IL-32β and IL-32γ isoforms was observed in HIV-related KS biopsy specimens compared with non–HIV-related KS and NST. The splicing ratio of the IL-32 isoforms showed IL-32γ as the highest expressed isoform, followed by IL-32β, in HIV-related KS cases compared with non–HIV-related KS and NST. Our data suggest a possible survival mechanism by the splicing of IL-32γ to IL-32β and also IL-6, IL-8, and CXCR1 signaling pathways to reverse the proapoptotic effect of the IL-32γ isoform, leading to tumor cell survival and thus favoring tumor progression. The intracellular proinflammatory mediator IL-32 is associated with tumor progression; however, the mechanisms remain unknown. We studied IL-32 mRNA expression as well as expression of other proinflammatory cytokines and mediators, including IL-1α, IL-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α, the proangiogenic and antiapoptotic enzyme cyclooxygenase-2, the IL-8 receptor C-X-C chemokine receptor (CXCR) 1, and the intracellular kinase focal adhesion kinase-1. The interaction of IL-32 expression with expression of IL-6, TNF-α, IL-8, and cyclooxygenase-2 was also investigated. Biopsy specimens of 11 HIV-related, 7 non–HIV-related Kaposi sarcoma (KS), and 7 normal skin tissues (NSTs) of Dutch origin were analyzed. RNA was isolated from the paraffin material, and gene expression levels of IL-32 α, β, and γ isoforms, IL1a, IL1b, IL6, IL8, TNFA, PTGS2, CXCR1, and PTK2 were determined using real-time quantitative PCR. Significantly higher expression of IL-32β and IL-32γ isoforms was observed in HIV-related KS biopsy specimens compared with non–HIV-related KS and NST. The splicing ratio of the IL-32 isoforms showed IL-32γ as the highest expressed isoform, followed by IL-32β, in HIV-related KS cases compared with non–HIV-related KS and NST. Our data suggest a possible survival mechanism by the splicing of IL-32γ to IL-32β and also IL-6, IL-8, and CXCR1 signaling pathways to reverse the proapoptotic effect of the IL-32γ isoform, leading to tumor cell survival and thus favoring tumor progression. IL-32 is a proinflammatory and proapoptotic cytokine that plays a role in carcinogenesis,1Nishida A. Andoh A. Inatomi O. Fujiyama Y. Interleukin-32 expression in the pancreas.J Biol Chem. 2009; 284: 17868-17876Crossref PubMed Scopus (86) Google Scholar, 2Seo E.H. Kang J. Kim K.H. Cho M.C. Lee S. Kim H.J. Kim J.H. Kim E.J. Park D.K. Kim S.H. Choi Y.K. Kim J.M. Hong J.T. Yoon D.Y. Detection of expressed IL-32 in human stomach cancer using ELISA and immunostaining.J Microbiol Biotechnol. 2008; 18: 1606-1612PubMed Google Scholar, 3Plantinga T.S. Costantini I. Heinhuis B. Huijbers A. Semango G. Kusters B. Netea M.G. Hermus A.R. Smit J.W. Dinarello C.A. Joosten L.A. Netea-Maier R.T. A promoter polymorphism in human interleukin-32 modulates its expression and influences the risk and the outcome of epithelial cell-derived thyroid carcinoma.Carcinogenesis. 2013; 34: 1529-1535Crossref PubMed Scopus (30) Google Scholar inflammation,4Dinarello C.A. Kim S.H. IL-32, a novel cytokine with a possible role in disease.Ann Rheum Dis. 2006; 65 Suppl 3: iii61-iii64PubMed Google Scholar and host defense to infectious agents, such as HIV and Mycobacterium tuberculosis.5Netea M.G. Azam T. Lewis E.C. Joosten L.A. Wang M. Langenberg D. Meng X. Chan E.D. Yoon D.Y. Ottenhoff T. Kim S.H. Dinarello C.A. Mycobacterium tuberculosis induces interleukin-32 production through a caspase-1/IL-18/interferon-gamma-dependent mechanism.PLoS Med. 2006; 3: e277Crossref PubMed Scopus (162) Google Scholar, 6Nold M.F. Nold-Petry C.A. Pott G.B. Zepp J.A. Saavedra M.T. Kim S.H. Dinarello C.A. Endogenous IL-32 controls cytokine and HIV-1 production.J Immunol. 2008; 181: 557-565Crossref PubMed Scopus (104) Google Scholar IL-32 mRNA is preferentially expressed in immune cells.7Kim S.H. Han S.Y. Azam T. Yoon D.Y. Dinarello C.A. Interleukin-32: a cytokine and inducer of TNFalpha.Immunity. 2005; 22: 131-142PubMed Scopus (492) Google Scholar IL-32 induces production of proinflammatory cytokines, including tumor necrosis factor (TNF)-α, IL-1β, IL-6, and IL-8 via NF-κB, p38 mitogen-activated protein kinase, and activating protein-1 activation.7Kim S.H. Han S.Y. Azam T. Yoon D.Y. Dinarello C.A. Interleukin-32: a cytokine and inducer of TNFalpha.Immunity. 2005; 22: 131-142PubMed Scopus (492) Google Scholar The sources of IL-32 include natural killer cells, T cells, monocytes, endothelial cells, and epithelial cells,8Nold-Petry C.A. Nold M.F. Zepp J.A. Kim S.H. Voelkel N.F. Dinarello C.A. IL-32-dependent effects of IL-1beta on endothelial cell functions.Proc Natl Acad Sci U S A. 2009; 106: 3883-3888Crossref PubMed Scopus (104) Google Scholar, 9Heinhuis B. Popa C.D. van Tits B.L. Kim S.H. Zeeuwen P.L. van den Berg W.B. van der Meer J.W. van der Vliet J.A. Stalenhoef A.F. Dinarello C.A. Netea M.G. Joosten L.A. Towards a role of interleukin-32 in atherosclerosis.Cytokine. 2013; 64: 433-440Crossref PubMed Scopus (32) Google Scholar with endothelial cells reported to exhibit the highest expression. Human IL-32 exists as six splice variants (α, β, γ, δ, ε, and ζ).7Kim S.H. Han S.Y. Azam T. Yoon D.Y. Dinarello C.A. Interleukin-32: a cytokine and inducer of TNFalpha.Immunity. 2005; 22: 131-142PubMed Scopus (492) Google Scholar The most frequently found isoforms, however, are IL-32 α, β, and γ .7Kim S.H. Han S.Y. Azam T. Yoon D.Y. Dinarello C.A. Interleukin-32: a cytokine and inducer of TNFalpha.Immunity. 2005; 22: 131-142PubMed Scopus (492) Google Scholar The IL-32 isoforms probably originate by splicing of pre-mRNA of the isoform IL-32γ.10Joosten L.A. Heinhuis B. Netea M.G. Dinarello C.A. Novel insights into the biology of interleukin-32.Cell Mol Life Sci. 2013; 70: 3883-3892Crossref PubMed Scopus (75) Google Scholar IL-32γ is regarded as the most active isoform of the cytokine, whereas splicing of IL-32γ into IL-32β is thought to be a safety switch in controlling the effects of IL-32γ and thereby reducing chronic inflammation.11Heinhuis B. Koenders M.I. van de Loo F.A. Netea M.G. van den Berg W.B. Joosten L.A. Inflammation-dependent secretion and splicing of IL-32{gamma} in rheumatoid arthritis.Proc Natl Acad Sci U S A. 2011; 108: 4962-4967Crossref PubMed Scopus (89) Google Scholar IL-32 is being extensively studied and has been implicated in several diseases, ranging from autoimmune disorders (eg, rheumatoid arthritis),12Joosten L.A. Netea M.G. Kim S.H. Yoon D.Y. Oppers-Walgreen B. Radstake T.R. Barrera P. van de Loo F.A. Dinarello C.A. van den Berg W.B. IL-32, a proinflammatory cytokine in rheumatoid arthritis.Proc Natl Acad Sci U S A. 2006; 103: 3298-3303Crossref PubMed Scopus (286) Google Scholar to chronic obstructive pulmonary disease,13Calabrese F. Baraldo S. Bazzan E. Lunardi F. Rea F. Maestrelli P. Turato G. Lokar-Oliani K. Papi A. Zuin R. Sfriso P. Balestro E. Dinarello C.A. Saetta M. IL-32, a novel proinflammatory cytokine in chronic obstructive pulmonary disease.Am J Respir Crit Care Med. 2008; 178: 894-901Crossref PubMed Scopus (128) Google Scholar and to infectious diseases (eg, HIV).6Nold M.F. Nold-Petry C.A. Pott G.B. Zepp J.A. Saavedra M.T. Kim S.H. Dinarello C.A. Endogenous IL-32 controls cytokine and HIV-1 production.J Immunol. 2008; 181: 557-565Crossref PubMed Scopus (104) Google Scholar More recently, it has been implicated in carcinogenesis.1Nishida A. Andoh A. Inatomi O. Fujiyama Y. Interleukin-32 expression in the pancreas.J Biol Chem. 2009; 284: 17868-17876Crossref PubMed Scopus (86) Google Scholar, 2Seo E.H. Kang J. Kim K.H. Cho M.C. Lee S. Kim H.J. Kim J.H. Kim E.J. Park D.K. Kim S.H. Choi Y.K. Kim J.M. Hong J.T. Yoon D.Y. Detection of expressed IL-32 in human stomach cancer using ELISA and immunostaining.J Microbiol Biotechnol. 2008; 18: 1606-1612PubMed Google Scholar Carcinogenesis and inflammation are heavily intertwined processes, and the interplay between these two processes is influenced by a large array of inflammatory mediators, including IL-32. Lin and Karin,14Lin W.W. Karin M. A cytokine-mediated link between innate immunity, inflammation, and cancer.J Clin Invest. 2007; 117: 1175-1183Crossref PubMed Scopus (1516) Google Scholar in 2007, described several cytokines involved in inflammatory and/or immune diseases associated with cancer development. They described the involvement of cytokines, such as TNF-α and IL-6. TNF-α was identified as the major host factor that enhances the growth of lung metastases in a mouse model, in part through activation of NF-κB in the tumor cells. IL-6, on the other hand, has been documented to be involved in the progression of Kaposi sarcoma (KS).15Zhang Y.J. Bonaparte R.S. Patel D. Stein D.A. Iversen P.L. Blockade of viral interleukin-6 expression of Kaposi's sarcoma-associated herpesvirus.Mol Cancer Ther. 2008; 7: 712-720Crossref PubMed Scopus (21) Google Scholar Both of these cytokines are induced by IL-32, as previously demonstrated by Kim et al.7Kim S.H. Han S.Y. Azam T. Yoon D.Y. Dinarello C.A. Interleukin-32: a cytokine and inducer of TNFalpha.Immunity. 2005; 22: 131-142PubMed Scopus (492) Google Scholar This function of IL-32 illustrates its importance in carcinogenesis. KS is a multifocal tumor that manifests in four distinct epidemiological forms: AIDS-associated KS, iatrogenic KS, endemic KS, and African KS. KS-associated herpes virus (KSHV; alias human herpes virus-8) is the causative agent of KS.16Chang Y. Cesarman E. Pessin M.S. Lee F. Culpepper J. Knowles D.M. Moore P.S. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma.Science. 1994; 266: 1865-1869Crossref PubMed Scopus (4973) Google Scholar The virus causes formation of KS spindle cells of the endothelial cell lineage; their derivation is still uncertain (ie, whether they are blood vessel or lymphatic vessel endothelial cells).17Sturzl M. Zietz C. Monini P. Ensoli B. Human herpesvirus-8 and Kaposi's sarcoma: relationship with the multistep concept of tumorigenesis.Adv Cancer Res. 2001; 81: 125-159Crossref PubMed Scopus (63) Google Scholar In KS tumors, almost 80% of the KS spindle cells are infected with KSHV,18Sturzl M. Blasig C. Schreier A. Neipel F. Hohenadl C. Cornali E. Ascherl G. Esser S. Brockmeyer N.H. Ekman M. Kaaya E.E. Tschachler E. Biberfeld P. Expression of HHV-8 latency-associated T0.7 RNA in spindle cells and endothelial cells of AIDS-associated, classical and African Kaposi's sarcoma.Int J Cancer. 1997; 72: 68-71Crossref PubMed Scopus (153) Google Scholar most of which are infected latently with the virus. During this latency, only a few proteins are expressed, which function primarily in maintenance of the viral genome, cellular proliferation, and activation of NF-κB and p38 mitogen-activated protein kinase signaling cascades. Recent studies have proposed cyclooxygenase-2 (COX-2) and its metabolite prostaglandin E2 as two pivotal proinflammatory/oncogenic molecules to play a role in the expression of major KSHV latency-associated nuclear antigen-1.19Sharma-Walia N. Paul A.G. Bottero V. Sadagopan S. Veettil M.V. Kerur N. Chandran B. Kaposi's sarcoma associated herpes virus (KSHV) induced COX-2: a key factor in latency, inflammation, angiogenesis, cell survival and invasion.PLoS Pathog. 2010; 6: e1000777Crossref PubMed Scopus (110) Google Scholar COX-2, an enzyme involved in prostanoid synthesis, has been heavily associated with KSHV latent infection and, thus, KS tumor progression.19Sharma-Walia N. Paul A.G. Bottero V. Sadagopan S. Veettil M.V. Kerur N. Chandran B. Kaposi's sarcoma associated herpes virus (KSHV) induced COX-2: a key factor in latency, inflammation, angiogenesis, cell survival and invasion.PLoS Pathog. 2010; 6: e1000777Crossref PubMed Scopus (110) Google Scholar This proangiogenic and apoptotic enzyme is up-regulated by mitogenic and inflammatory stimuli and serves to modulate the immune system in favor of KS progression. Sharma-Walia et al19Sharma-Walia N. Paul A.G. Bottero V. Sadagopan S. Veettil M.V. Kerur N. Chandran B. Kaposi's sarcoma associated herpes virus (KSHV) induced COX-2: a key factor in latency, inflammation, angiogenesis, cell survival and invasion.PLoS Pathog. 2010; 6: e1000777Crossref PubMed Scopus (110) Google Scholar demonstrated the great potential of tumor cell death induction by specific inhibition of COX-2. Interestingly, IL-32 expression has been shown to be induced by COX-2 stimulation in cervical cancer, and it has also been recently reported to be proangiogenic.20Lee S. Kim J.H. Kim H. Kang J.W. Kim S.H. Yang Y. Kim J. Park J. Park S. Hong J. Yoon D.Y. Activation of the interleukin-32 pro-inflammatory pathway in response to human papillomavirus infection and over-expression of interleukin-32 controls the expression of the human papillomavirus oncogene.Immunology. 2011; 132: 410-420Crossref PubMed Scopus (49) Google Scholar, 21Nold-Petry C.A. Rudloff I. Baumer Y. Ruvo M. Marasco D. Botti P. Farkas L. Cho S.X. Zepp J.A. Azam T. Dinkel H. Palmer B.E. Boisvert W.A. Cool C.D. Taraseviciene-Stewart L. Heinhuis B. Joosten L.A. Dinarello C.A. Voelkel N.F. Nold M.F. IL-32 promotes angiogenesis.J Immunol. 2014; 192: 589-602Crossref PubMed Scopus (66) Google Scholar This interaction is a key factor in the mechanisms of immune evasion and progression of KS. Furthermore, KSHV is documented to express a viral IL-6 protein homologous to human IL-6. Zhang et al15Zhang Y.J. Bonaparte R.S. Patel D. Stein D.A. Iversen P.L. Blockade of viral interleukin-6 expression of Kaposi's sarcoma-associated herpesvirus.Mol Cancer Ther. 2008; 7: 712-720Crossref PubMed Scopus (21) Google Scholar demonstrated, in their study, that blockade of this viral IL-6 has an effect on tumor progression. Both COX-2 and IL-32 are reported to influence IL-6 expression.19Sharma-Walia N. Paul A.G. Bottero V. Sadagopan S. Veettil M.V. Kerur N. Chandran B. Kaposi's sarcoma associated herpes virus (KSHV) induced COX-2: a key factor in latency, inflammation, angiogenesis, cell survival and invasion.PLoS Pathog. 2010; 6: e1000777Crossref PubMed Scopus (110) Google Scholar, 22Jung M.Y. Son M.H. Kim S.H. Cho D. Kim T.S. IL-32gamma induces the maturation of dendritic cells with Th1- and Th17-polarizing ability through enhanced IL-12 and IL-6 production.J Immunol. 2011; 186: 6848-6859Crossref PubMed Scopus (64) Google Scholar Several studies have reported IL-32 overexpression in tumor cells compared with normal cells, indicating that IL-32 is associated with carcinogenesis.1Nishida A. Andoh A. Inatomi O. Fujiyama Y. Interleukin-32 expression in the pancreas.J Biol Chem. 2009; 284: 17868-17876Crossref PubMed Scopus (86) Google Scholar, 2Seo E.H. Kang J. Kim K.H. Cho M.C. Lee S. Kim H.J. Kim J.H. Kim E.J. Park D.K. Kim S.H. Choi Y.K. Kim J.M. Hong J.T. Yoon D.Y. Detection of expressed IL-32 in human stomach cancer using ELISA and immunostaining.J Microbiol Biotechnol. 2008; 18: 1606-1612PubMed Google Scholar On the other hand, Oh et al23Oh J.H. Cho M.C. Kim J.H. Lee S.Y. Kim H.J. Park E.S. Ban J.O. Kang J.W. Lee D.H. Shim J.H. Han S.B. Moon D.C. Park Y.H. Yu D.Y. Kim J.M. Kim S.H. Yoon D.Y. Hong J.T. IL-32gamma inhibits cancer cell growth through inactivation of NF-kappaB and STAT3 signals.Oncogene. 2011; 30: 3345-3359Crossref PubMed Scopus (81) Google Scholar demonstrated that IL-32γ inhibits tumor growth by inhibiting expression of NF-κB and STAT3. Similarly, Yun et al24Yun H.M. Oh J.H. Shim J.H. Ban J.O. Park K.R. Kim J.H. Lee D.H. Kang J.W. Park Y.H. Yu D. Kim Y. Han S.B. Yoon D.Y. Hong J.T. Antitumor activity of IL-32beta through the activation of lymphocytes, and the inactivation of NF-kappaB and STAT3 signals.Cell Death Dis. 2013; 4: e640Crossref PubMed Scopus (48) Google Scholar demonstrated that IL-32β inhibits tumor growth by increasing cytotoxic lymphocyte numbers and by inactivating the NF-κB and STAT3 pathways through modulation of cytokine levels in tumor tissues. These effects may be influenced by the differential role of IL-32 between cell types and its participation in different intracellular pathways, as described previously.10Joosten L.A. Heinhuis B. Netea M.G. Dinarello C.A. Novel insights into the biology of interleukin-32.Cell Mol Life Sci. 2013; 70: 3883-3892Crossref PubMed Scopus (75) Google Scholar We have recently demonstrated that individuals bearing the IL-32 genetic variant rs28372698, which leads to increased IL-32γ gene expression and higher production of proinflammatory cytokines, have a higher risk for developing epithelial cell–derived thyroid carcinoma.3Plantinga T.S. Costantini I. Heinhuis B. Huijbers A. Semango G. Kusters B. Netea M.G. Hermus A.R. Smit J.W. Dinarello C.A. Joosten L.A. Netea-Maier R.T. A promoter polymorphism in human interleukin-32 modulates its expression and influences the risk and the outcome of epithelial cell-derived thyroid carcinoma.Carcinogenesis. 2013; 34: 1529-1535Crossref PubMed Scopus (30) Google Scholar Moreover, these individuals require higher dosages of radioactive iodide, the standard therapy after thyroidectomy, to achieve successful tumor remission.3Plantinga T.S. Costantini I. Heinhuis B. Huijbers A. Semango G. Kusters B. Netea M.G. Hermus A.R. Smit J.W. Dinarello C.A. Joosten L.A. Netea-Maier R.T. A promoter polymorphism in human interleukin-32 modulates its expression and influences the risk and the outcome of epithelial cell-derived thyroid carcinoma.Carcinogenesis. 2013; 34: 1529-1535Crossref PubMed Scopus (30) Google Scholar Depending on the function of the isoforms, IL-32 has thus far been demonstrated to play a critical role in both tumor progression and inhibition. It has been demonstrated to be overexpressed in tumors, with the IL-32β and IL-32γ isoforms being the most abundant. However, the mechanism behind the tumor progression has not been studied and explained. The IL-32γ isoform has predominantly a proapoptotic action and, thus, induces cell death. Nold et al6Nold M.F. Nold-Petry C.A. Pott G.B. Zepp J.A. Saavedra M.T. Kim S.H. Dinarello C.A. Endogenous IL-32 controls cytokine and HIV-1 production.J Immunol. 2008; 181: 557-565Crossref PubMed Scopus (104) Google Scholar demonstrated the potency of IL-32 to induce HIV-infected cells to undergo apoptosis after IL-32 up-regulation. They also demonstrated that on reduction of endogenous IL-32, HIV viral load tremendously increased in these cells compared with cells expressing IL-32.6Nold M.F. Nold-Petry C.A. Pott G.B. Zepp J.A. Saavedra M.T. Kim S.H. Dinarello C.A. Endogenous IL-32 controls cytokine and HIV-1 production.J Immunol. 2008; 181: 557-565Crossref PubMed Scopus (104) Google Scholar Nevertheless, contrary to this effect, tumor cells have been shown to progress and not to undergo apoptosis, even on high expression of IL-32. Splicing of the IL-32γ isoform into IL-32β has been described before to occur in THP1 cells, which was demonstrated to be relevant within the pathogenesis and severity of rheumatoid arthritis.11Heinhuis B. Koenders M.I. van de Loo F.A. Netea M.G. van den Berg W.B. Joosten L.A. Inflammation-dependent secretion and splicing of IL-32{gamma} in rheumatoid arthritis.Proc Natl Acad Sci U S A. 2011; 108: 4962-4967Crossref PubMed Scopus (89) Google Scholar These studies have revealed that the splicing from IL-32γ into IL-32β may be regarded as a safety switch because IL-32β is a less potent proinflammatory mediator than IL-32γ, leading to decreased production of proinflammatory cytokines, such as IL-1β and IL-6. We hypothesized that this splicing mechanism of IL-32γ to IL-32β is a survival mechanism that inhibits tumor cells from going into apoptosis, even with high expression of IL-32γ. In the present study, the role of the survival mechanism that tumor cells might use to survive high expression levels of IL-32γ, which is predominantly proinflammatory and proapoptotic, was studied. The IL-32 splicing pattern in KS was studied to determine its influence on tumor progression. Furthermore, the effect of IL-6, IL-8, TNF-α, COX-2, C-X-C chemokine receptor (CXCR) 1, and focal adhesion kinase (FAK)-1 on tumor progression and apoptosis induction was investigated. With these experiments, we determined the possible interplay between IL-32 and COX-2 and show their interaction in the initiation of tumor survival pathways in KS. Understanding these interactions may be a step forward toward development of an immune-modulating IL-32–based therapy for KS and other endothelial cell pathologies driven by chronic infections. Patient tissues of HIV-infected KS cases (N = 11) and non–HIV-infected KS cases (N = 7) from patients of Dutch origin, confirmed by histology and immunohistochemistry, were selected from the database of the Department of Pathology, Radboud University Medical Center (Nijmegen, the Netherlands). These samples were selected on the basis of sample availability in the pathology department archives as well as quality and quantity of RNA obtained. Samples with poor quality and/or low RNA yield were excluded from analysis. Patients' personal details were decoded to maintain confidentiality by giving a study number to all formalin-fixed, paraffin-embedded tissue biopsy specimens. Ethical approval was obtained for all patient tissues. Per block, five sections (20 μm thick) were cut from formalin-fixed, paraffin-embedded material under semisterile conditions and transferred to a sterile tube. RNA was isolated by lysis and RNA precipitation protocols. Tissue samples were disrupted and homogenized by an overnight incubation with Proteinase K (Qiagen, Valencia, CA). RNA extraction was performed by using RNA-Bee, according to the manufacturer's protocol (AMS Biotechnology, Abingdon, UK), including chloroform phase separation and isopropanol precipitation. Isolated RNA was subsequently transcribed into cDNA by using random hexamers (Promega, Leiden, the Netherlands), followed by real-time quantitative PCR (qPCR) using the SYBR Green method (Applied Biosystems, Foster City, CA). The following primers were used for detection: IL-32α, 5′-GCTGGAGGACGACTTCAAAGA-3′ (forward) and 5′-GGGCTCCGTAGGACTTGTCA-3′ (reverse); IL-32β, 5′-CAGTGGAGCTGGGTCATCTCA-3′ (forward) and 5′-GGGCCTTCAGCTTCTTCATGTCATCA-3′ (reverse); IL-32γ, 5′-AGGCCCGAATGGTAATGCT-3′ (forward) and 5′-CCACAGTGTCCTCAGTGTCACA-3′ (reverse); TNF-α, 5′-GACGTGGAAGTGGCAGAAGAG-3′ (forward) and 5′-TGCCACAAGCAGGAATGAGA-3′ (reverse); IL-8, 5-AAGAGAGCTCTGTCTGGACC-3′ (forward) and 5′-GATATTCTCTTGGCCCTTGG-3′ (reverse); IL-6, 5′-GGTACATCCTCGACGGCATCT-3′ (forward) and 5′-GTGCCTCTTTGCTGCTTTCAC-3′ (reverse); COX-2, 5′-GGTCTGGTGCCTGGTCTGATGATG-3′ (forward) and 5′-GTCCTTTCAAGGAGAATGGTGC-3′ (reverse); CXCR1, 5′-CGACTGTGGGCGGATTCTTG-3′ (forward) and 5′-AGACCGATACCATGTGCTCT-3′ (reverse); and FAK-1, 5′-TCCCTATGGTGAAGGAAGT-3′ (forward) and 5′-TTCTGTGCCATCTCAATCT-3′ (reverse). Data were corrected for expression of the housekeeping gene β2-microglobulin, for which the primers 5′-ATGAGTATGCCTGCCGTGTG-3′ (forward) and 5′-CCAAATGCGGCATCTTCAAAC-3′ (reverse) were used. Gene expression values were calculated by using the comparative threshold cycle (CT) method. The CT data for the different genes and the housekeeping gene β2-microglobulin were used to generate CT values (ΔCT = CT target gene − CT housekeeping gene). Thereafter, the relative quantity was calculated by 2ΔCt × 1000. Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords from healthy donors after obtaining informed consent. Cells were cultured in RPMI 1640 medium (Gibco-Invitrogen, Gaithersburg, MD) supplemented with penicillin/streptomycin, glutamine, pyruvate, heat-inactivated pooled human serum (10%), and heat-inactivated fetal bovine serum (10%). HUVECs were cultured in 0.2% w/v gelatinized (Sigma-Aldrich, St. Louis, MO) tissue flasks/plates (Corning Inc., Corning, NY) at 37°C and 5% CO2. HUVECs were stimulated with 50 μg/mL poly(I:C) (Invivogen, Toulouse, France) or in serum-free RPMI 1640 medium. Twenty-four hours after stimulation, total RNA was isolated by adding Tri-reagent (Sigma-Aldrich) to the cells and processed, as described by Heinhuis et al.25Heinhuis B. Koenders M.I. van de Loo F.A. van Lent P.L. Kim S.H. Dinarello C.A. Joosten L.A. van den Berg W.B. IL-32gamma and Streptococcus pyogenes cell wall fragments synergise for IL-1-dependent destructive arthritis via upregulation of TLR-2 and NOD2.Ann Rheum Dis. 2010; 69: 1866-1872Crossref PubMed Scopus (28) Google Scholar Finally, IL-32 mRNA expression was assessed by qPCR. IL-32 protein expression was evaluated by immunohistochemical staining of formalin-fixed, paraffin-embedded KS tissue sections. To remove the paraffin, tissues were incubated twice in xylene and successively in 100%, 96%, and 70% alcohol for 5 minutes each step. Antigens were retrieved with citrate buffer for 2 minutes in the microwave (800 W) and 10 minutes at room temperature citrate buffer: pH = 6.0, 16.4 mL sodium citrate (0.1 mol/L) with 3.6 mL citric acid (0.1 mol/L) in 180 mL H2O. The endogenous peroxidase activity was blocked with 3% H2O2 in methanol for 15 minutes at room temperature. Furthermore, because tumor-like tissues contain endogenous biotin, this was blocked in the tissue sections by an avidin/biotin blocking kit, according to the manufacturer's protocol (Vector Laboratories, Burlingame, CA). Sections were incubated with 20% goat serum diluted in phosphate-buffered saline (PBS) for 10 minutes and subsequently with the first antibody (polyclonal goat anti-human IL-32 AF3040 antibody or goat polyclonal IgG isotype control AB-108-C; R&D Systems, Minneapolis, MN), both 2.5 μg/mL in PBS supplemented with 5% goat serum overnight at room temperature. After washing with PBS, sections were incubated with the second antibody (rabbit anti–goat-BIOT Vector BA-5000; Vector Laboratories), 1:500 diluted in PBS supplemented with 5% rabbit serum, for 30 minutes at room temperature. The ABC–horseradish peroxidase complex (ABCkit-HRP Vector PK-6101; Vector Laboratories), 1:200 diluted in PBS, was applied to the sections for 30 minutes at room temperature. The substrate solution was added for 5 minutes at room temperature: 0.5 mL of diaminobenzidene in 9.5 mL of PBS and 10 μL of H2O2. Tissues were counterstained with hematoxylin for 30 seconds at room temperature. Slides were dehydrated with consecutive incubation in 70%, 96%, and 100% alcohol and xylene (two times) for 5 minutes each step. Sections were mounted in Permount (Thermo Fisher Scientific, Waltham, MA). To determine significant differences between the test samples against the controls, U-tests were used to determine the two-tailed P values. To account for the comparisons of normal skin tissue (NST) versus HIV+ KS+ and NST versus HIV− KS+, a two-tailed P < 0.025 was considered statistically significant. The analyses were performed using GraphPad Prism version 5 (GraphPad Software, Inc., La Jolla, CA). The general characteristics of the data are listed in Table 1.Table 1The General Characteristics of the DataCytokineHIV+, KS+NSTHIV−, KS+NRangeMedian (IQR)P valueNRangeMedian (IQR)NRangeMedian (IQR)P valueIL-32β1116.18–174.9643.39 (23.9–107.12)0.002660.42–32.6812.66 (3.49–14.92)512.62–100.1524.20 (17.47–43.39)0.0679IL-32γ1111.29–92.6336.15 (21.35–60.11)0.02761.98–31.2518.19 (9.24–20.85)58.92–80.5023.47 (12.67–27.35)0.47IL-870.16–1135.626.33 (0.84–821.69)0.9452.45–27.934.37 (3.95–4.65)50.16–5.30.48 (0.48–0.64)0.08CXCR1810.15–215.2544.94 (28.52–143.53)0.2065.18–51.0620.79 (15.52–47.04)50.80–137.703.93 (2.57–41.32)0.36TNF-α111.67–90.2217.32 (7.98–27.41)0.7940.57–232.9913.07 (2.48–127.38)52.55–72.254.12 (2.81–6.72)0.62FAK-180.00–31.420.50 (0.16–2.23)0.3130.06–0.250.17 (0.06–0.25)50.3–2.150.74 (0.37–0.92)0.025IL-651.40–772.1016.22 (3.59–26.31)0.3660.00–65.368.29 (0.00–20.21)53.56–9.806.00 (5.80–8.78)1.00IL-1α50.09–17.161.10 (0.13–13.74)0.7550.10–1.860.89 (0.58–1.26)40.16–0.870.53 (0.21–0.83)0.33IL-1β30.95–29.084.92 (0.09–29.08)0.2520.03–1.210.62 (0.03–1.21)40.89–0.490.36 (0.19–0.46)1.00COX-282.19–216.7113.19 (2.65–46.98)0.6657.78–71.2211.31 (10.63–14.60)50.88–17.792.92 (2.42–12.58)0.25IL-32γ/β110.26–3.880.72 (0.34–0.96)0.00960.96–4.731.60 (1.16–2.65)50.37–2.170.72 (0.54–0.80)0.04+, Present; −, absent; COX, cyclooxygenase; CXCR, C-X-C chemokine receptor; FAK, focal adhesion kinase; IQR, interquartile range; KS, Kaposi sarcoma; NST, normal skin tissue; TNF, tumor necrosis factor. Open table in a new tab +, Present; −, absent; COX, cyclooxygenase; CXCR, C-X-C chemokine receptor; FAK, focal adhesion kinase; IQR, interquartile range; KS, Kaposi sarcoma; NST, normal skin tissue; TNF, tumor necrosis factor. To investigate whether IL-32 is expressed in KS, qPCR was used. Expression of IL-32 isoforms was observed in KS cases (Figure 1), with abundant quantities of IL-32β and IL-32γ isoforms in particular. IL-32α was not detected in KS and NST (data not shown). IL-32β showed significantly elevated expression levels in HIV-related KS patients compared with NST (Figure 1A). HIV-related KS cases also showed marginally statistically significant high levels of IL-32γ compared with normal skin controls (Figure 1B). To assess IL-32 protein levels, immunohistochemical staining was performed for IL-32 in HIV-related KS. Cases with the highest and lowest levels of IL-32 mRNA were selected. High IL-32 protein expression was observed in HIV-related KS compared with isotype control. Corresponding high levels of IL-32 protein were detected in high IL-32 mRNA expressing tissues. Also, hematoxylin and eosin staining of HIV-related KS and non–HIV–related KS was performed, and the degree of inflammation in the same cases was assessed. More cellularity and increased numbers of lymphocytes and plasma cells were observed in non-HIV KS (Figure 2). To investigate the role of other selected proinflammatory cytokines and mediators in the tumor progression and to test our hypothesis of COX-2 and IL-8 involvement, the mRNA expression levels of these cytokines were determined by qPCR. FAK-1 (Figure 3H) showed significantly increased expression in non–HIV-related KS compared with NST. IL-8 (Figure 3A), CXCR1 (Figure 3B), IL-6 (Figure 3C), IL-1α (Figure 3D), IL-1β (Figure 3E), and COX-2 (Figure 3F) showed higher expression in HIV-related KS compared with non–HIV-related KS and NST. TNF-α (Figure 3G) showed lower expression in HIV- and non–HIV-related KS compared with NST. To investigate the relative expression of IL-32 in KS compared with normal expression in uninfected skin tissue and virus-infected epithelial cells, qPCR was conducted on poly(I:C) stimulated HUVECs. To investigate the relative quantities of the different IL-32 isoforms, the ratio of IL-32γ/IL-32β was calculated. The IL-32γ/IL-32β (Figure 4B) ratio was statistically significantly increased in HUVECs stimulated with poly(I:C) compared with medium control. The splicing ratio in HIV-related KS was significantly reduced compared with NST (Figure 4A). We propose IL-32 plays a role in carcinogenesis through the induction of IL-8 production. IL-8 is then excreted to the extracellular environment, where it interacts with its receptor CXCR1 and initiates the IL-8 signaling pathway, which occurs through FAK-1 as its downstream signaling molecule. In turn, this signaling pathway of IL-8 plays a role in tumor survival by production of cell survival proteins. KS seems to be using this mechanism, because increased IL-32γ, IL-32β, IL-8, CXCR1, and FAK-1 expression was observed. Interestingly, KS showed a decreased IL-32γ/IL-32β splicing ratio compared with NST, whereas poly(I:C) stimulated HUVECs showed an increased IL-32γ/IL-32β splicing ratio. This observation is interesting and indicates that human herpes virus-8–induced KS behaves differently from other viral infectious agents, which usually lead to increased IL-32γ/IL-32β splicing ratios. We propose IL-32 plays a role in carcinogenesis through the splicing of the proapoptotic IL-32γ isoform to its lesser potent isoform IL-32β and eventually to the IL-32α isoform. We propose that this splicing mechanism plays an important role in the balance between apoptosis and survival among other mechanisms. The model in Figure 5 shows the interaction between IL-32 and IL-8. Furthermore, the key mechanisms and mediators that play a role in the balance between apoptosis and cell survival with respect to IL-32 involvement are shown. In this study, elevated expression of IL-32γ and IL-32β isoforms was observed in HIV-related KS cases. The current data show that IL-32γ and IL-32β isoforms are significantly elevated in HIV-related KS. This trend has been observed in other tumors by other investigators as well2Seo E.H. Kang J. Kim K.H. Cho M.C. Lee S. Kim H.J. Kim J.H. Kim E.J. Park D.K. Kim S.H. Choi Y.K. Kim J.M. Hong J.T. Yoon D.Y. Detection of expressed IL-32 in human stomach cancer using ELISA and immunostaining.J Microbiol Biotechnol. 2008; 18: 1606-1612PubMed Google Scholar, 23Oh J.H. Cho M.C. Kim J.H. Lee S.Y. Kim H.J. Park E.S. Ban J.O. Kang J.W. Lee D.H. Shim J.H. Han S.B. Moon D.C. Park Y.H. Yu D.Y. Kim J.M. Kim S.H. Yoon D.Y. Hong J.T. IL-32gamma inhibits cancer cell growth through inactivation of NF-kappaB and STAT3 signals.Oncogene. 2011; 30: 3345-3359Crossref PubMed Scopus (81) Google Scholar and is further supported by the present study. In addition to this observation, our data also show the occurrence of splicing activity, especially from IL-32γ to IL-32β. Heinhuis et al26Heinhuis B. Koenders M.I. van den Berg W.B. Netea M.G. Dinarello C.A. Joosten L.A. Interleukin 32 (IL-32) contains a typical alpha-helix bundle structure that resembles focal adhesion targeting region of focal adhesion kinase-1.J Biol Chem. 2012; 287: 5733-5743Crossref PubMed Scopus (63) Google Scholar demonstrated, in a human embryonic kidney cell line, that cells undergo apoptosis on transiently elevated expression of IL-32γ and IL-32β. In addition to that, Goda et al27Goda C. Kanaji T. Kanaji S. Tanaka G. Arima K. Ohno S. Izuhara K. Involvement of IL-32 in activation-induced cell death in T cells.Int Immunol. 2006; 18: 233-240Crossref PubMed Scopus (159) Google Scholar indicated that IL-32 may be involved in activation-induced cell death in T cells. However, the KS tumor cells do not undergo apoptosis, even with elevated expression of these proapoptotic and proinflammatory IL-32 isoforms demonstrated to induce cell death. We, thus, speculate that an additional pathway is favored by the proapoptotic and proinflammatory microenvironment. A possible role for IL-8 in this survival mechanism was observed that may be used by these tumor cells to progress in an IL-32γ and IL-32β environment. We speculate that KS tumor cells, despite having high IL-32γ to IL-32β splicing activity and, thus, high expression of IL-32γ and IL-32β isoforms, can survive by up-regulating IL-8 and CXCR1 expression. This serves as the survival mechanism by increasing FAK-1 expression and, thus, triggers cell survival mechanisms. The induction of IL-8 production by IL-32 expression has been demonstrated before by other investigators.7Kim S.H. Han S.Y. Azam T. Yoon D.Y. Dinarello C.A. Interleukin-32: a cytokine and inducer of TNFalpha.Immunity. 2005; 22: 131-142PubMed Scopus (492) Google Scholar HIV-related KS cases showed significantly elevated expression of IL-32β and IL-32γ compared with NST. This observation fits well with findings in other tumor types, as discussed in the first paragraph. The elevated levels of the proapoptotic and proinflammatory IL-32 isoforms in HIV- and non–HIV-related KS tumors may be explained by the fact that KSHV-8 is involved with or without HIV, respectively. These KSHV-8 viruses induce inflammation through immune activation. This leads to increased production of proinflammatory cytokines, including IL-32γ and IL-32β. Nold et al6Nold M.F. Nold-Petry C.A. Pott G.B. Zepp J.A. Saavedra M.T. Kim S.H. Dinarello C.A. Endogenous IL-32 controls cytokine and HIV-1 production.J Immunol. 2008; 181: 557-565Crossref PubMed Scopus (104) Google Scholar demonstrated mechanistically the antiviral effect of IL-32 toward HIV-1 and proposed that IL-32 is a natural inhibitor of the virus. The KSHV-8 virus, on the other hand, uses viral IL-6, a viral analog of human IL-6, which is induced by IL-32 to stimulate cells and, thus, bind to gp130, contributing to the pathogenic processes involved.15Zhang Y.J. Bonaparte R.S. Patel D. Stein D.A. Iversen P.L. Blockade of viral interleukin-6 expression of Kaposi's sarcoma-associated herpesvirus.Mol Cancer Ther. 2008; 7: 712-720Crossref PubMed Scopus (21) Google Scholar HIV-related KS tumors were observed to exhibit a significantly higher IL-32γ to IL-32β splicing activity. This splicing activity was much more efficient than in NST. Furthermore, HIV-related KS showed a trend toward increased expression of IL-1α, IL-1β, IL-8, IL-6, CXCR1, and COX-2. Taken together, these observations suggest KS as an efficient tumor in using the two proposed survival mechanisms. These data further indicate that tumor cells may use splicing activity of IL-32γ to IL-32β and up-regulation of IL-8 and IL-6 signaling. This leads us to further speculate on the involvement of IL-8 and IL-6 signaling in the survival mechanism of tumor cells, thus favoring tumor progression. TNF-α and FAK-1 also showed a tendency of decreased expression in HIV-related KS compared with NST and non–HIV-related KS. This tendency is worth exploring further. We can speculate the tendency to be because of the initial levels of IL-32γ and IL-32β. The high splicing activity of IL-32γ to IL-32β in HIV-related KS may explain the decreased tendency of TNF-α expression compared with non–HIV-related KS and NST because the IL-32β isoform is a less potent proinflammatory cytokine compared with the IL-32γ isoform. The current study has several limitations, mainly the lack of cell culture experiments using KS cell lines to explicitly study the proposed mechanisms separately. Nevertheless, this is the first study, to our knowledge, to explore these pathways in KS tumor progression. In conclusion, our findings bring into perspective key matters regarding KS tumor cell survival. HIV- and non–HIV-related KS tumors show efficient IL-32γ to IL-32β splicing activity compared with NST. We speculate that the splicing of IL-32γ to IL-32β serves as one of the survival mechanisms used by KS cells. Furthermore, the trend toward increased IL-8, IL-6, IL-1α, IL-1β, CXCR1, and COX-2 expression also seems to be following the same trend of being most evident in KS. We propose further studies into these novel pathways of elevated IL-8 signaling and IL-32 splicing that tumor cells may be using for their survival, because these findings may serve as an important cornerstone for cancer therapy in the future.
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