The Two Faces of Immune-Related lncRNAs in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a group of cancers originating from the mucosal epithelium in the oral cavity, larynx, oropharynx, nasopharynx, and hypopharynx. Molecular factors can be key in the diagnosis, prognosis, and treatment of HNSCC patients. Long non-coding RNAs (lncRNAs) are molecular regulators composed of 200 to 100,000 nucleotides that act on the modulation of genes that activate signaling pathways associated with oncogenic processes such as proliferation, migration, invasion, and metastasis in tumor cells. However, up until now, few studies have discussed the participation of lncRNAs in modeling the tumor microenvironment (TME) to generate a protumor or antitumor environment. Nevertheless, some immune-related lncRNAs have clinical relevance, since AL139158.2, AL031985.3, AC104794.2, AC099343.3, AL357519.1, SBDSP1, AS1AC108010.1, and TM4SF19-AS1 have been associated with overall survival (OS). MANCR is also related to poor OS and disease-specific survival. MiR31HG, TM4SF19-AS1, and LINC01123 are associated with poor prognosis. Meanwhile, LINC02195 and TRG-AS1 overexpression is associated with favorable prognosis. Moreover, ANRIL lncRNA induces resistance to cisplatin by inhibiting apoptosis. A superior understanding of the molecular mechanisms of lncRNAs that modify the characteristics of TME could contribute to increasing the efficacy of immunotherapy.

E6/E7 and E6* From HPV16 and HPV18 Upregulate IL-6 Expression Independently of p53 in Keratinocytes.

Keratinocyte infection with high-risk human papillomavirus genotypes has been linked to cancer development. In cervix, the alpha HPV16 and HPV18 have been reported as the mayor causative agents of cervical cancer. Oncogenic progression and chronic inflammation are closely related processes, with IL-6 as one of the main pro-inflammatory cytokines involved. However, there are limited studies about the regulation of IL-6 by low and high risk HPVs and the HPV proteins implicated in this modulation. In this work, we report the overexpression of IL-6 in HPV infected cervical cancer derived cell lines (HeLa and SiHa) compared to non-tumorigenic keratinocytes (HaCaT), and in Cervical Intraepithelial Neoplasia grade 1 HPV16 and HPV18 positive cervical samples compared to HPV negative samples without lesions. Moreover, we generated HaCaT keratinocytes that express E5, E6, and E7 from high risk (16 or 18) or low risk (62 and 84) HPVs. E5 proteins do not modify IL-6 expression, while E7 modestly increase it. Interestingly, E6 proteins in HaCaT cells upregulate IL-6 mRNA expression and protein secretion. Indeed, in HaCaT cells that express high risk HPV16E6 or HPV18E6 proteins, only the truncated E6* isoforms were expressed, showing the stronger IL-6 overexpression, while in HaCaT cells that express low risk HPV62 and HPV84 full length E6 proteins, IL-6 was also upregulated but not so drastically. Since HaCaT cells have a mutated p53 form that is not degraded by the introduction of E6 or E6/E7, it seems that E6/E7 regulate IL-6 by an additional mechanism independent of p53. In addition, basal keratinocytes showed a strong expression of IL-6R using immunohistochemistry, suggesting an autocrine mechanism over proliferative cells. Altogether, IL-6 cytokine expression in keratinocytes is upregulated by E6 and E7 proteins from HPVs 16, 18, 62, and 84, especially by high risk HPV16 and HPV18 E6*, which may contribute to promote a pro-inflammatory and highly proliferative microenvironment that can persist over time and lead to cervical tumorigenesis.

GPER Overexpression in Cervical Cancer Versus Premalignant Lesions: Its Activation Induces Different Forms of Cell Death.

BACKGROUND: The effect of estrogen has been traditionally studied through the modulation of its alpha and beta nuclear receptors; however, the G Protein-Coupled Estrogen Receptor (GPER) has been recently involved in the pathology of numerous tumors. Although the study of GPER in cervical cancer has begun, its contribution still remains to be completely evaluated. OBJECTIVE: The purpose of this work was to determine the expression of this receptor in different degrees of cervical lesions and whether the stimulation with its specific agonist (G-1) modulated mechanisms of cell survival or cell death in cervical cancer cells. METHODS: Sections of 44 formalin-fixed paraffin-embedded blocks from patients were analyzed by automated immunohistochemistry. After the stimulation with G-1, proliferation was evaluated by the xCELLigence technology, the integrity of the mitochondrial membrane permeability by MitoCaptureTM fluorescence staining, apoptosis by flow cytometry, and senescence by the senescence-associated ß-galactosidase kit. RESULTS: GPER was widely expressed in cervical cancer but not in its precursor lesions. The staining was predominantly cytoplasmic, although it was also important in the nucleus of the epithelial cells. G-1 inhibited proliferation, decreased the mitochondrial permeability, and increased the percentage of apoptosis in SiHa, HeLa, and C-33A. Only in C-33A, an increase of the cells in necrosis was observed, whereas SiHa was the only cell line in which senescence was evidenced. CONCLUSION: GPER is a receptor associated with cervical cancer that inhibits the growth and induces different mechanisms of death in cells derived from uterine cervical cancer. It suggests that GPER can be considered a pharmacological target that prevents the development of cervical carcinogenesis.

IL10 haplotypes are associated with diabetic nephropathy susceptibility in patients from western Mexico.

INTRODUCTION: Diabetic Nephropathy (DN) is the main cause of chronic kidney disease (CKD) in diabetic patients. An IL-10 imbalance could be related to renal hypertrophy and trigger to nephropathy. Three promoter polymorphisms (-1082G>A, -819C>T, and -592C>A) at IL10 gene have been associated with changes in the IL-10 expression and DN susceptibility. Therefore, the aim of this study was to analyze this association in Mexican patients with DN. METHODS: We conducted a case-control study on 128 patients with DN and 150 control subjects (CS) from western Mexico. All patients were tested for IL10 polymorphisms by PCR-RFLP. Allele frequencies, genotypes, and haplotypes were compared between groups. The significant haplotypes were correlated with patient clinical features. RESULTS: IL10 gene ATC haplotype (-1082A/-819T/-592C) was found significantly more frequent in DN patients than in CS (P < 0.001; OR = 3.6, 95% CI: 1.7-7.4). Similarly GTA (-1082G/-819T/-592A) haplotype was more frequent in DN patients than CS with significant differences (P < 0.05; OR = 4.02, 95% CI: 1.10-14.78). There were no correlations between IL10 haplotypes and clinical parameters in patients with DN. However, that there is a trend of higher serum urea levels and lower eGFR in ATC haplotype carriers compared to carriers of the other haplotypes (P < 0.05). CONCLUSIONS: These results indicate that IL10 promoter haplotypes ATC and GTA carriers have a higher risk factor to develop DN in the western Mexican population.

Association of extrapituitary prolactin promoter polymorphism with disease susceptibility and anti-RNP antibodies in Mexican patients with systemic lupus erythematosus.

Introduction: Prolactin (PRL) is a 23-kDa protein that can be synthesized and secreted by pituitary and extrapituitary tissues such as immune cells due to its expression being regulated by two independent promoter regions. The promoter which is responsible for extrapituitary expression contains the single nucleotide polymorphism (SNP) -1149 G/T previously associated with autoimmune diseases in various populations. This study evaluates the relationship of PRL -1149 G/T polymorphism with PRL serum levels and clinical characteristics in systemic lupus erythematosus (SLE) patients from western Mexico. Material and methods: One hundred and sixty-three SLE patients classified according to the 1982 American College of Rheumatology (ACR) SLE classification criteria and 326 unrelated control subjects (CS), both from western Mexico, were included. The PRL -1149 G/T polymorphism was genotyped using the polymerase chain reaction restriction fragment length polymorphism technique, and both PRL serum levels and autoantibodies were measured by enzyme-linked immunosorbent assay (ELISA). Results: We found an association between the PRL -1149 TT genotype and SLE according to the recessive genetic model (OR = 2.26, 95% CI: 1.01-5.08, p = 0.04). The TT genotype was associated with anti-RNP antibodies (p = 0.04) and with higher scores of the Mex-SLEDAI (p = 0.02). Moreover, SLE patients showed elevated PRL serum levels (12.4 ng/ml; p < 0.01), and this condition was associated with renal activity and the presence of anti-RNP antibodies. Conclusions: PRL -1149 TT genotype is associated with susceptibility to SLE in a Mexican-Mestizo population, and high PRL serum levels are associated with anti-RNP antibodies and renal activity.

Expression of ADAM10, Fas, FasL and Soluble FasL in Patients with Oral Squamous Cell Carcinoma (OSCC) and their Association with Clinical-Pathological Parameters.

ADAM10 has been implicated in the progression of various solid tumors. ADAM10 regulates the cleavage of the FasL ectodomain from the plasma membrane of different cell types, generating the soluble FasL fragment (sFasL). Currently, there are few studies in oral squamous cell carcinoma (OSCC) that correlate levels of ADAM10 and FasL in the tumor microenvironment with clinical parameters of the disease. To determine the expression of ADAM10, Fas, FasL and sFasL in patients with OSCC and its association with TNM stage. Twenty-five patients with OSCC and 25 healthy controls were included. Biopsies of tumor tissue from patients with OSCC and buccal mucosa in controls were obtained. ADAM10, Fas, and FasL were analyzed by Western blotting. sFasL was quantified by ELISA. ADAM10 and Fas decreased significantly in OSCC compared with controls. Relatedly, within the OSCC group, Fas and ADAM10 decreased in accordance with tumor disease stage; in stages I/II, as well as in tumors of smaller diameter (T1-T2), ADAM10 showed higher levels when compared to patients with T3-T4 tumors and in stage III-IV. FasL in the tumor microenvironment and serum FasL showed no significant differences between both groups. Levels of complete FasL and cleaved FasL were positively correlated in controls; this correlation is preserved in patients with tumors in early stages (I-II), but is lost in later stage (III-IV). The dysregulation of ADAM10, Fas and FasL could be useful indicators of the progression and severity of OSCC.

Liver fibrosis in bile duct-ligated rats correlates with increased hepatic IL-17 and TGF-ß2 expression.

UNLABELLED: BACKGROUND AND RATIONALE FOR THE STUDY: IL-17, TGF-ß1/2 are cytokines involved in the development of kidney, pulmonary and liver fibrosis. However, their expression kinetics in the pathogenesis of cholestatic liver fibrosis have not yet been fully explored. The aim of the study was to analyze the expression of IL-17, RORγt, NKp46, TGF-ß1, and TGF-ß2 in the liver of rats with bile duct ligation (BDL). RESULTS: Hepatic IL-17A gene expression analyzed by qRT-PCR showed a dramatic increase of 350 and 10 fold, at 8 and 30 days post BDL, respectively. TGFß1 and TGFß2 gene expression significantly increased throughout the whole fibrotic process. At the protein level in liver homogenates, IL-17, TGF-ß1, and RORγt significantly increased at 8 and 30 days after BDL. Interestingly, a significant increase in the protein levels of TGF-ß2 and decrease of NKp46 was observed only 30 days after BDL. Unexpectedly, TGF-ß2 exhibited stronger signals than TGF-ß1 at the gene expression and protein levels. Histological analysis showed bile duct proliferation and collagen deposition. CONCLUSIONS: Our results suggest that pro-fibrogenic cytokines IL-17, TGF-ß1 and, strikingly, TGF-ß2 might be important players of liver damage in the pathogenesis of early and advanced experimental cholestatic fibrosis. Th17 cells might represent an important source of IL-17, while NK cell depletion may account for the perpetuation of liver damage in the BDL model.

Expression of autocrine prolactin and the short isoform of prolactin receptor are associated with inflammatory response and apoptosis in monocytes stimulated with Mycobacterium bovis proteins.

Increased levels of prolactin (PRL) have recently been associated with carcinogenesis and the exacerbation of autoimmune diseases, and might be involved in the progression of tuberculosis (TB). To investigate the relationship between PRL and prolactin receptor (PRLr) expression with inflammatory response and apoptosis in monocytes, we used THP-1 cells stimulated with antigens of the Mycobacterium bovis AN5 strain culture filtrate protein (CFP-M. bovis). Western blot (WB), real-time Polymerase chain reaction (PCR), and immunocytochemistry were performed to identify both PRL and PRLr molecules. PRL bioactivity and proinflammatory cytokine detection were assessed. The results showed that PRL and PRLr messenger RNA (mRNA) were synthesized in THP-1 monocytes induced with CFP-M. bovis at peaks of 176- and 404-fold, respectively. PRL forms of 60 and 80kDa and PRLr isoforms of 40, 50, and 65kDa were also identified as time-dependent, while 60-kDa PRL, as well as 40-, and 50-kDa PRLr, were found as soluble forms in culture media and later in the nucleus of THP-1 monocytes. PRL of 60kDa released by monocytes exhibited bioactivity in Nb2 cells, and both synthesized PRL and synthesized PRLr were related with nitrite and proinflammatory cytokine levels proapoptotic activity in CFP-M. bovis-induced monocytes. Our results suggest the overexpression of a full-autocrine loop of PRL and PRLr in monocytes that enhances the inflammatory response and apoptosis after priming with M. bovis antigens.

Substantial increase in the frequency of circulating CD4+NKG2D+ T cells in patients with cervical intraepithelial neoplasia grade 1.

BACKGROUND: The NKG2D receptor confers important activating signals to NK cells via ligands expressed during cellular stress and viral infection. This receptor has generated great interest because not only is it expressed on NK cells, but it is also seen in virtually all CD8+ cytotoxic T cells and is classically considered absent in CD4+ T cells. However, recent studies have identified a distinctive population of CD4+ T cells that do express NKG2D, which could represent a particular cytotoxic effector population involved in viral infections and chronic diseases. On the other hand, increased incidence of human papillomavirus-associated lesions in CD4+ T cell-immunocompromised individuals suggests that CD4+ T cells play a key role in controlling the viral infection. Therefore, this study was focused on identifying the frequency of NKG2D-expressing CD4+ T cells in patients with cervical intraepithelial neoplasia (CIN) 1. Additionally, factors influencing CD4+NKG2D+ T cell expansion were also measured. RESULTS: Close to 50% of patients with CIN 1 contained at least one of the 37 HPV types detected by our genotyping system. A tendency for increased CD4+ T cells and CD8+ T cells and decreased NK cells was found in CIN 1 patients. The percentage of circulating CD4+ T cells co-expressing the NKG2D receptor significantly increased in women with CIN 1 versus control group. Interestingly, the increase of CD4+NKG2D+ T cells was seen in patients with CIN 1, despite the overall levels of CD4+ T cells did not significantly increase. We also found a significant increase of soluble MICB in CIN 1 patients; however, no correlation with the presence of CD4+NKG2D+ T cells was seen. While TGF-beta was significantly decreased in the group of CIN 1 patients, both TNF-alpha and IL-15 showed a tendency to increase in this group. CONCLUSIONS: Taken together, our results suggest that the significant increase within the CD4+NKG2D+ T cell population in CIN 1 patients might be the result of a chronic exposure to viral and/or pro-inflammatory factors, and concomitantly might also influence the clearance of CIN 1-type lesion.

Lipopolysaccharide induces the expression of an autocrine prolactin loop enhancing inflammatory response in monocytes.

BACKGROUND: Prolactin from pituitary gland helps maintain homeostasis but it is also released in immune cells where its function is not completely understood. Pleiotropic functions of prolactin (PRL) might be mediated by different isoforms of its receptor (PRLr). METHODS: The aim of this study was to investigate the relationship between the eventual synthesis of PRL and PRLr isoforms with the inflammatory response in monocytes. We used THP-1 and monocytes isolated from healthy subjects stimulated with lipopolysaccharide (LPS). Western blot, real time PCR and immunocytochemistry were performed to identify both molecules. The bioactivity of the PRL was assessed using a bioassay and ELISA to detect pro inflammatory cytokines. RESULTS: PRLr mRNA and PRL mRNA were synthesized in THP-1 monocytes activated with LPS with peaks of 300-fold and 130-fold, respectively. The long (100 kDa) and the intermediate (50 kDa) isoforms of PRLr and big PRL (60 kDa) were time-dependent upregulated for monocytes stimulated with LPS. This expression was confirmed in monocytes from healthy subjects. The PRLr intermediate isoform and the big PRL were found soluble in the culture media and later in the nucleus in THP-1 monocytes stimulated with LPS. Big PRL released by monocytes showed bioactivity in Nb2 Cells, and both PRL and PRLr, synthesized by monocytes were related with levels of nitrites and proinflammatory citokines. CONCLUSIONS: Our results suggest the expression of a full-autocrine loop of PRL enhances the inflammatory response in activated monocytes. This response mediated by big PRL may contribute to the eradication of potential pathogens during innate immune response in monocytes but may also contribute to inflammatory disorders.

Increased expression of the prolactin receptor is associated with malignant laryngeal tumors.

The altered expression of the prolactin receptor (PRLR) has been associated with the development of various types of cancer, particularly breast, prostate and endometrial cancer. However, in laryngeal tumors, the expression of PRLR has not yet been documented. The aim of this study was to determine the expression and localization of PRLR in laryngeal cancer (LC) in comparison with recurrent respiratory papillomatosis (RRP). PRLR expression was analyzed in 48 paraffin-embedded tissues (18 RRP and 30 laryngeal cancer tissues) by immunoperoxidase staining. Furthermore, PRLR expression was evaluated in ten samples from each group by Western blot analysis and quantitative real-time PCR. PRLR was observed in all laryngeal tumors at different intensities. PRLR overexpression was significantly associated (P<0.005) with LC. The staining pattern was homogeneous, mainly cytoplasmic, and confined to the tumor area. We found increased expression of different isoforms in LC in comparison with RRP. Our results suggest a possible role of PRL/PRLR in the development of LC. PRLR may be useful as a target for further investigations in laryngeal tissues.

Cervical cancer cell lines expressing NKG2D-ligands are able to down-modulate the NKG2D receptor on NKL cells with functional implications.

BACKGROUND: Cervical cancer represents the third most commonly diagnosed cancer and the fourth leading cause of cancer-related deaths in women worldwide. Natural killer (NK) cells play an important role in the defense against viruses, intracellular bacteria and tumors. NKG2D, an activating receptor on NK cells, recognizes MHC class I chain-related molecules, such as MICA/B and members of the ULBP/RAET1 family. Tumor-derived soluble NKG2D-ligands have been shown to down-modulate the expression of NKG2D on NK cells. In addition to the down-modulation induced by soluble NKG2D-ligands, it has recently been described that persistent cell-cell contact can also down-modulate NKG2D expression. The goal of this study was to determine whether the NKG2D receptor is down-modulated by cell-cell contact with cervical cancer cells and whether this down-modulation might be associated with changes in NK cell activity. RESULTS: We demonstrate that NKG2D expressed on NKL cells is down-modulated by direct cell contact with cervical cancer cell lines HeLa, SiHa, and C33A, but not with non-tumorigenic keratinocytes (HaCaT). Moreover, this down-modulation had functional implications. We found expression of NKG2D-ligands in all cervical cancer cell lines, but the patterns of ligand distribution were different in each cell line. Cervical cancer cell lines co-cultured with NKL cells or fresh NK cells induced a marked diminution of NKG2D expression on NKL cells. Additionally, the cytotoxic activity of NKL cells against K562 targets was compromised after co-culture with HeLa and SiHa cells, while co-culture with C33A increased the cytotoxic activity of the NKL cells. CONCLUSIONS: Our results suggest that differential expression of NKG2D-ligands in cervical cancer cell lines might be associated with the down-modulation of NKG2D, as well as with changes in the cytotoxic activity of NKL cells after cell-cell contact with the tumor cells.