Overexpression of the HIF hydroxylases PHD1, PHD2, PHD3 and FIH are individually and collectively unfavorable prognosticators for NSCLC survival

Introduction

Hypoxia induced factors (HIFs) are at the heart of the adaptive mechanisms cancer cells must implement for survival. HIFs are regulated by four hydroxylases; Prolyl hydroxylase (PHD)-1,-2,-3 and factor inhibiting HIF (FIH). We aimed to investigate the prognostic impact of these oxygen sensors in NSCLC.

Methods

Tumor tissue samples from 335 resected stages I to IIIA NSCLC patients was obtained and tissue microarrays (TMAs) were constructed. Hydroxylase expression was evaluated by immunohistochemistry.

Principal Findings

There was scorable expression for all HIF hydroxylases in tumor cells, but not in stroma. In univariate analyses, high tumor cell expression of all the HIF hydroxylases were unfavorable prognosticators for disease-specific survival (DSS); PHD1 (P = 0.023), PHD2 (P = 0.013), PHD3 (P = 0.018) and FIH (P = 0.033). In the multivariate analyses we found high tumor cell expression of PHD2 (HR = 2.03, CI 95% 1.20–3.42, P = 0.008) and PHD1 (HR = 1.45, CI 95% 1.01–2.10, P = 0.047) to be significant independent prognosticators for DSS. Besides, there was an additive prognostic effect by the increasing number of highly expressed HIF hydroxylases. Provided none high expression HIF hydroxylases, the 5-year survival was 80% vs. 23% if all four were highly expressed (HR = 6.48, CI 95% 2.23–18.8, P = 0.001).

Conclusions

HIF hydroxylases are, in general, poor prognosticators for NSCLC survival. PHD1 and PHD2 are independent negative prognostic factors in NSCLC. Moreover, there is an additive poor prognostic impact by an increasing number of highly expressed HIF hydroxylases.     

Activated TLR signaling in atherosclerosis among women with lower Framingham risk score: the multi-ethnic study of atherosclerosis

Background

Atherosclerosis is the leading cause of cardiovascular disease (CVD). Traditional risk factors can be used to identify individuals at high risk for developing CVD and are generally associated with the extent of atherosclerosis; however, substantial numbers of individuals at low or intermediate risk still develop atherosclerosis.

Results

A case-control study was performed using microarray gene expression profiling of peripheral blood from 119 healthy women in the Multi-Ethnic Study of Atherosclerosis cohort aged 50 or above. All participants had low (<10%) to intermediate (10% to 20%) predicted Framingham risk; cases (N = 48) had coronary artery calcium (CAC) score >100 and carotid intima-media thickness (IMT) >1.0 mm, whereas controls (N = 71) had CAC<10 and IMT <0.65 mm. We identified two major expression profiles significantly associated with significant atherosclerosis (odds ratio 4.85; P<0.001); among those with Framingham risk score <10%, the odds ratio was 5.30 (P<0.001). Ontology analysis of the gene signature reveals activation of a major innate immune pathway, toll-like receptors and IL-1R signaling, in individuals with significant atherosclerosis.

Conclusion

Gene expression profiles of peripheral blood may be a useful tool to identify individuals with significant burden of atherosclerosis, even among those with low predicted risk by clinical factors. Furthermore, our data suggest an intimate connection between atherosclerosis and the innate immune system and inflammation via TLR signaling in lower risk individuals.     

Deprived TLR9 Expression in Apparently Healthy Nasal Mucosa Might Trigger Polyp-Growth in Chronic Rhinosinusitis Patients

Background

The origin of nasal polyps in chronic rhinosinusitis is unknown, but the role of viral infections in polyp growth is clinically well established. Toll-like receptors (TLRs) have recently emerged as key players in our local airway defense against microbes. Among these, TLR9 has gained special interest in viral diseases. Many studies on chronic rhinosinusitis with nasal polyps (CRSwNP) compare polyp tissue with nasal mucosa from polyp-free individuals. Knowledge about changes in the turbinate tissue bordering the polyp tissue is limited.

Objectives

To analyse the role of TLR9 mediated microbial defense in tissue bordering the polyp.

Methods

Nasal polyps and turbinate tissue from 11 patients with CRSwNP and turbinate tissue from 11 healthy controls in total were used. Five biopsies from either group were analysed immediately with flow cytometry regarding receptor expression and 6 biopsies were used for in vitro stimulation with a TLR9 agonist, CpG. Cytokine release was analysed using Luminex. Eight patients with CRSwNP in total were intranasally challenged with CpG/placebo 24 hours before surgery and the biopsies were collected and analysed as above.

Results

TLR9 expression was detected on turbinate epithelial cells from healthy controls and polyp epithelial cells from patients, whereas TLR9 was absent in turbinate epithelial cells from patients. CpG stimulation increased the percentage cells expressing TLR9 and decreased percentage cells expressing VEGFR2 in turbinate tissue from patients. After CpG stimulation the elevated levels of IL-6, G-CSF and MIP-1β in the turbinate tissue from patients were reduced towards the levels demonstrated in healthy controls.

Conclusion

Defects in the TLR9 mediated microbial defense in the mucosa adjacent to the anatomic origin of the polyp might explain virus induced polyp growth. CpG stimulation decreased VEGFR2, suggesting a role for CpG in polyp formation. The focus on turbinate tissue in patients with CRSwNP opens new perspectives in CRSwNP-research.     

Prognostic impacts of angiopoietins in NSCLC tumor cells and stroma: VEGF-A impact is strongly associated with Ang-2

Introduction

Angiopoietins and their receptor Tie-2 are, in concert with VEGF-A, key mediators in angiogenesis. This study evaluates the prognostic impact of all known human angiopoietins (Ang-1, Ang-2 and Ang-4) and their receptor Tie-2, as well as their relation to the prognostic expression of VEGF-A.

Methods

335 unselected stage I-IIIA NSCLC-patients were included and tissue samples of respective tumor cells and stroma were collected in tissue microarrays (TMAs). Immunohistochemistry (IHC) was used to semiquantitatively evaluate the expression of markers in duplicate tumor and stroma cores.

Principal Findings

In univariate analyses, low tumor cell expression of Ang-4 (P = 0.046) and low stromal expressions of Ang-4 (P = 0.009) and Ang-2 (P = 0.017) were individually associated with a poor survival. In the multivariate analysis, low stromal Ang-2 (HR 1.88; CI 95% 1.15-3.08) and Ang-4 (HR 1.47, CI 95% 1.02–2.11, P = 0.04) expressions were independently associated with a poor prognosis. In patients with high tumor cell expression of Ang-2, a concomitantly high tumor VEGF-A expression mediated a dramatic survival reduction (P<0.001). In the multivariate analysis of patients with high Ang-2 expression, high tumor VEGF-A expression appeared an independent poor prognosticator (HR 6.43; CI 95% 2.46–16.8; P<0.001).

Conclusions

In tumor cells, only Ang-4 expression has prognostic impact in NSCLC. In tumor stroma, Ang-4 and Ang-2 are independently associated with survival. The prognostic impact of tumor cell VEGF-A in NSCLC appears strongly associated with a concomitantly high tumor cell expression of Ang-2.     

The prognostic impact of TGF-β1, fascin, NF-κB and PKC-ζ expression in soft tissue sarcomas

Aims

Transforming growth factor-β (TGF-β), fascin, nuclear factor-kappa B (NF-κB) p105, protein-kinase C-zeta (PKC-ζ), partioning-defective protein-6 (Par-6), E-cadherin and vimentin are tumor promoting molecules through mechanisms involved in cell dedifferentiation. In soft tissue sarcomas, their expression profile is poorly defined and their significance is uncertain. We aimed to investigate the prognostic impact of TGF-β1, NF-κB p105, PKC-ζ, Par-6α, E-cadherin and vimentin in non-gastrointestinal stromal tumor soft tissue sarcomas (non-GIST STSs).

Patients and Methods

Tumor samples and clinical data from 249 patients with non-GIST STS were obtained, and tissue microarrays (TMAs) were constructed for each specimen. Immunohistochemistry (IHC) was used to evaluate marker expression in tumor cells.

Results

In univariate analysis, the expression levels of TGF-β1 (P = 0.016), fascin (P = 0.006), NF-κB p105 (P = 0.022) and PKC-ζ, (P = 0.042) were significant indicators for disease specific survival (DSS). In the multivariate analysis, high TGF-β1 expression was an independent negative prognostic factor for DSS (HR = 1.6, 95% CI = 1.1–2.4, P = 0.019) in addition to tumor depth, malignancy grade, metastasis at diagnosis, surgery and positive resection margins.

Conclusion

Expression of TGF-β1 was significantly associated with aggressive behavior and shorter DSS in non-GIST STSs.     

Protein phosphatase 2A mediates dormancy of glioblastoma multiforme-derived tumor stem-like cells during hypoxia

Purpose

The hypoxic microenvironment of glioblastoma multiforme (GBM) is thought to increase resistance to cancer therapies. Recent evidence suggests that hypoxia induces protein phosphatase 2A (PP2A), a regulator of cell cycle and cell death. The effects of PP2A on GBM tumor cell proliferation and survival during hypoxic conditions have not been studied.

Experimental Design

Expression of PP2A subunits and HIF-α proteins was measured in 65 high-grade astrocytoma and 18 non-neoplastic surgical brain specimens by western blotting. PP2A activity was measured by an immunoprecipitation assay. For in vitro experiments, GBM-derived tumor stem cell-like cells (TSCs) were exposed to severe hypoxia produced by either CoCl₂ or 1% O₂. PP2A activity was inhibited either by okadaic acid or by shRNA depletion of the PP2A C subunit. Effects of PP2A activity on cell cycle progression and cell survival during hypoxic conditions were assessed using flow cytometry.

Results

In our patient cohort, PP2A activity was positively correlated with HIF-1∝ protein expression (P = 0.002). Patients with PP2A activity levels above 160 pMP had significantly worse survival compared to patients with levels below this threshold (P = 0.002). PP2A activity was an independent predictor of survival on multivariable analysis (P = 0.009). In our in vitro experiments, we confirmed that severe hypoxia induces PP2A activity in TSCs 6 hours after onset of exposure. PP2A activity mediated G1/S phase growth inhibition and reduced cellular ATP consumption in hypoxic TSCs. Conversely, inhibition of PP2A activity led to increased cell proliferation, exhaustion of intracellular ATP, and accelerated P53-independent cell death of hypoxic TSCs.

Conclusions

Our results suggest that PP2A activity predicts poor survival in GBM. PP2A appears to reduce the metabolic demand of hypoxic TSCs and enhances tumor cell survival. Modulation of PP2A may be a potential target for cancer therapy.     

Muscle hypertrophy in prepubescent tennis players: a segmentation MRI study

Purpose

To asses if tennis at prepubertal age elicits the hypertrophy of dominant arm muscles.

Methods

The volume of the muscles of both arms was determined using magnetic resonance imaging (MRI) in 7 male prepubertal tennis players (TP) and 7 non-active control subjects (CG) (mean age 11.0±0.8 years, Tanner 1–2).

Results

TP had 13% greater total muscle volume in the dominant than in the contralateral arm. The magnitude of inter-arm asymmetry was greater in TP than in CG (13 vs 3%, P<0.001). The dominant arm of TP was 16% greater than the dominant arm of CG (P<0.01), whilst non-dominant arms had similar total muscle volumes in both groups (P = 0.25), after accounting for height as covariate. In TP, dominant deltoid (11%), forearm supinator (55%) and forearm flexors (21%) and extensors (25%) were hypertrophied compared to the contralateral arm (P<0.05). In CG, the dominant supinator muscle was bigger than its contralateral homonimous (63%, P<0.05).

Conclusions

Tennis at prepubertal age is associated with marked hypertrophy of the dominant arm, leading to a marked level of asymmetry (+13%), much greater than observed in non-active controls (+3%). Therefore, tennis particpation at prepubertal age is associated with increased muscle volumes in dominant compared to the non-dominant arm, likely due to selectively hypertrophy of the loaded muscles.     

Innate Immune Receptors in Human Airway Smooth Muscle Cells: Activation by TLR1/2, TLR3, TLR4, TLR7 and NOD1 Agonists

Background

Pattern-recognition receptors (PRRs), including Toll-like receptors (TLRs), NOD-like receptors (NLRs) and RIG-I-like receptors (RLRs), recognize microbial components and trigger a host defense response. Respiratory tract infections are common causes of asthma exacerbations, suggesting a role for PRRs in this process. The present study aimed to examine the expression and function of PRRs on human airway smooth muscle cells (HASMCs).

Methods

Expression of TLR, NLR and RLR mRNA and proteins was determined using real-time RT-PCR, flow cytometry and immunocytochemistry. The functional responses to ligand stimulation were investigated in terms of cytokine and chemokine release, cell surface marker expression, proliferation and proteins regulating the contractile state.

Results

HASMCs expressed functional TLR2, TLR3, TLR4, TLR7 and NOD1. Stimulation with the corresponding agonists Pam₃CSK₄, poly(I:C), LPS, R-837 and iE-DAP, respectively, induced IL-6, IL-8 and GM-CSF release and up-regulation of ICAM-1 and HLA-DR, while poly(I:C) also affected the release of eotaxin and RANTES. The proliferative response was slightly increased by LPS. Stimulation, most prominently with poly(I:C), down-regulated myosin light chain kinase and cysteinyl leukotriene 1 receptor expression and up-regulated β₂-adrenoceptor expression. No effects were seen for agonist to TLR2/6, TLR5, TLR8, TLR9, NOD2 or RIG-I/MDA-5.

Conclusion

Activation of TLR2, TLR3, TLR4, TLR7 and NOD1 favors a synthetic phenotype, characterized by an increased ability to release inflammatory mediators, acquire immunomodulatory properties by recruiting and interacting with other cells, and reduce the contractile state. The PRRs might therefore be of therapeutic use in the management of asthma and infection-induced disease exacerbations.     

Beneficial effects of alternate dietary regimen on liver inflammation, atherosclerosis and renal activation

Background

Alternate day calorie restriction (CR) has been shown to be almost as beneficial as daily CR. The question arises whether this concept is also applicable to alternating dietary composition.

Objective

To seek evidence that alternating high cholesterol (HC) - cholesterol-free (CON) Western diet can effectively diminish hepatic and renal inflammation and cardiovascular risk factors as compared with daily HC-supplemented Western diet.

Design

Four groups of ApoE*3Leiden mice, a humanized model for atherosclerosis, were subjected to different feeding treatments for 16 weeks. Mice were fed CON diet; CON diet with 1% w/w cholesterol (HC); alternate (ALT) diet regimen of CON (4 days) and HC (3 days); or CON diet supplemented with 0.43% (w/w) cholesterol (MC), with overall dietary cholesterol intake equal to ALT. Plasma was analyzed for cardiovascular risk factors, aorta for atherosclerotic lesion formation, and liver and kidney for inflammation.

Results

ALT diet but not MC was almost as effective as daily CON feeding in preventing disease development. Compared to HC, the ALT group showed 62% lower hepatic nuclear factor kappa B (NF-κB) activity (P<0.001), a reduction of the circulating inflammatory markers E-selectin (−20%; P<0.05), vascular cell adhesion molecule 1 (VCAM-1; −15%; P<0.05) and Serum Amyloid A (SAA; −31%; P<0.05), smaller atherosclerotic lesion sizes (−51%; 46497±10791 µm² vs. 94664±16470 µm²; P<0.05) and diminished renal expression of specific inflammation and activation markers (VCAM-1, −27%; P<0.05; monocyte chemotactic protein-1 (MCP-1); −37%; P<0.01).

Conclusion

Alternate HC-CON feeding reproduced most of the beneficial effects of daily cholesterol-free diet, including strongly diminished hepatic, vascular and renal activation and inflammation; also atherosclerosis was reduced by half as compared to HC, albeit still higher compared to the CON group.     

Detoxification center-based sampling missed a subgroup of higher risk drug users, a case from Guangdong, China

Background

Injection drug use remains among the most important HIV transmission risk in China. Representativeness of drug users sampled from detoxification centers is questionable. A respondent driven sampling survey was conducted to compare the results with those from the detoxification center in the same city.

Methods

In 2008, two independent surveys were conducted in Dongguan, China, one for community-based drug users using respondent driven sampling and the other for drug users in a compulsory detoxification center as routine sentinel surveillance. Demographic and behavioral information were collected using the same structured questionnaire. Intravenous blood samples were collected to measure antibodies to HIV-1, and syphilis.

Results

Compared to those 400 drug users recruited from the detoxification center, the 303 community-based drug users had higher HIV prevalence (14.7% versus 4.0%, P = 0.04), lower syphilis prevalence (4.7% versus 10.8%, P = 0.07), higher proportion of injection drug use (83.9% versus 60.2%, P = 0.01) and syringe sharing (47.8% versus 36.3%, P = 0.10), more likely to be separated (12.4% versus 3.8%, P = 0.01) and being migrants from Guangxi province (31.4% versus 18.0%, P = 0.09), more engaging in commercial sex (64.4% versus 52.5%, P = 0.04). HIV prevalence and rate of syringe sharing were consistently higher among drug users from Guangxi.

Conclusions

Detoxification center-based sampling missed a subgroup with higher HIV prevalence and higher rate of injection drug use. While detoxification center-based sampled can be used to monitor the trend of HIV prevalence and risk behaviors over time, periodic community-based sampling is still necessary to avoid possible systematic error in detoxification center-based samples.     

Maternal TLR4 and NOD2 gene variants, pro-inflammatory phenotype and susceptibility to early-onset preeclampsia and HELLP syndrome

Background

Altered maternal inflammatory responses play a role in the development of preeclampsia and the hemolysis, elevated liver enzymes and low platelets (HELLP) syndrome. We examined whether allelic variants of the innate immune receptors Toll-like receptor 4 (TLR4) and nucleotide-binding oligomerization domain 2 (NOD2), that impair the inflammatory response to endotoxin, are related to preeclampsia and HELLP syndrome.

Methods and Findings

We determined five common mutations in TLR4 (D299G and T399I) and NOD2 (R702W, G908R and L1007fs) in 340 primiparous women with a history of early-onset preeclampsia, of whom 177 women developed HELLP syndrome and in 113 women with a history of only uneventful pregnancies as controls. In addition, we assessed plasma levels of pro-inflammatory biomarkers C-reactive protein, interleukin-6, soluble intercellular adhesion molecule-1, fibrinogen and von Willebrand factor in a subset of 214 women included at least six months after delivery. After adjustment for maternal age and chronic hypertension, attenuating allelic variants of TLR4 were more common in women with a history of early-onset preeclampsia than in controls (OR 2.9 [95% CI 1.2–6.7]). Highest frequencies for TLR4 variants were observed in women who developed HELLP syndrome (adjusted OR 4.1 [95% CI 1.7–9.8]). In addition, high levels of interleukin-6 and fibrinogen were associated with a history of early-onset preeclampsia. Combined positivity for any of the TLR4 and NOD2 allelic variants and high levels of interleukin-6 was 6.9-fold more common in women with a history of early-onset preeclampsia (95% CI 2.1–23.2) compared to controls.

Conclusions

We observed an association of common TLR4 and NOD2 gene variants, and pro-inflammatory phenotype with a history of early-onset preeclampsia and HELLP syndrome. These findings suggest involvement of the maternal innate immune system in severe hypertensive disorders of pregnancy.     

Profiling of VEGFs and VEGFRs as prognostic factors in soft tissue sarcoma: VEGFR-3 is an independent predictor of poor prognosis

Background

In non-gastrointestinal stromal tumor soft tissue sarcoma (non-GIST STS) optimal treatment is surgery with wide resection margins. Vascular endothelial growth factors (VEGFs) and receptors (VEGFRs) are known to be key players in the initiation of angiogenesis and lymphangiogenesis. This study investigates the prognostic impact of VEGFs and VEGFRs in non-GIST STS with wide and non-wide resection margins.

Methods

Tumor samples from 249 patients with non-GIST STS were obtained and tissue microarrays were constructed for each specimen. Immunohistochemistry was used to evaluate the expressions of VEGF-A, -C and -D and VEGFR-1, -2 and -3.

Results

In the univariate analyses, VEGF-A (P = 0.040) in the total material, and VEGF-A (P = 0.018), VEGF-C (P = 0.025) and VEGFR-3 (P = 0.027) in the subgroup with wide resection margins, were significant negative prognostic indicators of disease-specific survival (DSS). In the multivariate analysis, high expression of VEGFR-3 (P = 0.042, HR = 1.907, 95% CI 1.024-3.549) was an independent significant negative prognostic marker for DSS among patients with wide resection margins.

Conclusion

VEGFR-3 is a strong and independent negative prognostic marker for non-GIST STSs with wide resection margins.     

Nuclear factor-κB-dependent epithelial to mesenchymal transition induced by HIF-1α activation in pancreatic cancer cells under hypoxic conditions

Background

Epithelial to mesenchymal transition (EMT) induced by hypoxia is one of the critical causes of treatment failure in different types of human cancers. NF-κB is closely involved in the progression of EMT. Compared with HIF-1α, the correlation between NF-κB and EMT during hypoxia has been less studied, and although the phenomenon was observed in the past, the molecular mechanisms involved remained unclear.

Methodology/Principal Findings

Here, we report that hypoxia or overexpression of hypoxia-inducible factor-1α (HIF-1α) promotes EMT in pancreatic cancer cells. On molecular or pharmacologic inhibition of NF-κB, hypoxic cells regained expression of E-cadherin, lost expression of N-cadherin, and attenuated their highly invasive and drug-resistant phenotype. Introducing a pcDNA3.0/HIF-1α into pancreatic cancer cells under normoxic conditions heightened NF-κB activity, phenocopying EMT effects produced by hypoxia. Conversely, inhibiting the heightened NF-κB activity in this setting attenuated the EMT phenotype.

Conclusions/Significance

These results suggest that hypoxia or overexpression of HIF-1α induces the EMT that is largely dependent on NF-κB in pancreatic cancer cells.     

Novel M tuberculosis antigen-specific T-cells are early markers of infection and disease progression

Background

Mycobacterium tuberculosis Region-of-Difference-1 gene products present opportunities for specific diagnosis of M. tuberculosis infection, yet immune responses to only two gene-products, Early Secretory Antigenic Target-6 (ESAT-6) and Culture Filtrate Protein-10 (CFP-10), have been comprehensively investigated.

Methods

T-cell responses to Rv3873, Rv3878 and Rv3879c were quantified by IFN-γ-enzyme-linked-immunospot (ELISpot) in 846 children with recent household tuberculosis exposure and correlated with kinetics of tuberculin skin test (TST) and ESAT-6/CFP-10-ELISpot conversion over six months and clinical outcome over two years.

Results

Responses to Rv3873, Rv3878, and Rv3879c were present in 20–25% of contacts at enrolment. Rv3873 and Rv3879c responses were associated with and preceded TST conversion (P = 0.02 and P = 0.04 respectively), identifying these antigens as early targets of cell-mediated immunity following M. tuberculosis exposure. Responses to Rv3873 were additionally associated with subsequent ESAT-6/CFP-10-ELISpot conversion (P = 0.04). Responses to Rv3873 and Rv3878 predicted progression to active disease (adjusted incidence rate ratio [95% CI] 3.06 [1.05,8.95; P = 0.04], and 3.32 [1.14,9.71; P = 0.03], respectively). Presence of a BCG-vaccination scar was associated with a 67% (P = 0.03) relative risk reduction for progression to active tuberculosis.

Conclusions

These RD1-derived antigens are early targets of cellular immunity following tuberculosis exposure and T-cells specific for these antigens predict progression to active tuberculosis suggesting diagnostic and prognostic utility.     

PI3K and ERK-induced Rac1 activation mediates hypoxia-induced HIF-1α expression in MCF-7 breast cancer cells

Background

Hypoxia-inducible factor 1 (HIF-1α) expression induced by hypoxia plays a critical role in promoting tumor angiogenesis and metastasis. However, the molecular mechanisms underlying the induction of HIF-1α in tumor cells remain unknown.

Methodology/Principal Findings

In this study, we reported that hypoxia could induce HIF-1α and VEGF expression accompanied by Rac1 activation in MCF-7 breast cancer cells. Blockade of Rac1 activation with ectopic expression of an inactive mutant form of Rac1 (T17N) or Rac1 siRNA downregulated hypoxia-induced HIF-1α and VEGF expression. Furthermore, Hypoxia increased PI3K and ERK signaling activity. Both PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and ERK inhibitor U0126 suppressed hypoxia-induced Rac1 activation as well as HIF-1α expression. Moreover, hypoxia treatment resulted in a remarkable production of reactive oxygen species (ROS). N-acetyl-L-cysteine, a scavenger of ROS, inhibited hypoxia-induced ROS generation, PI3K, ERK and Rac1 activation as well as HIF-1α expression.

Conclusions/Significance

Taken together, our study demonstrated that hypoxia-induced HIF-1α expression involves a cascade of signaling events including ROS generation, activation of PI3K and ERK signaling, and subsequent activation of Rac1.     

Involvement of CCR6/CCL20/IL-17 axis in NSCLC disease progression

Objectives

Autocrine and paracrine chemokine/chemokine receptor-based interactions promote non-small-cell-lung-cancer (NSCLC) carcinogenesis. CCL20/CCR6 interactions are involved in prostatic and colonic malignancy pathogenesis. The expression and function of CCL20/CCR6 and its related Th-17 type immune response in NSCLC is not yet defined. We sought to characterize the role of the CCL20/CCR6/IL-17 axis in NSCLC tumor growth.

Methods

A specialized histopathologist blindly assessed CCL20/CCR6 expression levels in 49 tissue samples of NSCLC patients operated in our department. Results were correlated to disease progression. Colony assays, ERK signaling and chemokine production were measured to assess cancer cell responsiveness to CCL20 and IL-17 stimulation.

Results

CCL20 was highly expressed in the majority (38/49, 77.5%) of tumor samples. Only a minority of samples (8/49, 16.5%) showed high CCR6 expression. High CCR6 expression was associated with a shorter disease-free survival (P = 0.008) and conferred a disease stage-independent 4.87-fold increased risk for disease recurrence (P = 0.0076, CI 95% 1.52–15.563). Cancerous cell colony-forming capacity was increased by CCL20 stimulation; this effect was dependent in part on ERK phosphorylation and signaling. IL-17 expression was detected in NSCLC; IL-17 potentiated the production of CCL20 by cancerous cells.

Conclusion

Our findings suggest that the CCL20/CCR6 axis promotes NSCLC disease progression. CCR6 is identified as a potential new prognostic marker and the CCL20/CCR6/IL-17 axis as a potential new therapeutic target. Larger scale studies are required to consolidate these observations.     

Gene silencing of Toll-like receptor 2 inhibits proliferation of human liver cancer cells and secretion of inflammatory cytokines

Background

Toll-like receptors (TLRs) are key factors in the innate immune system and initiate the inflammatory response to foreign pathogens such as bacteria, fungi and viruses. In the microenvironment of tumorigenesis, TLRs can promote inflammation and cell survival. Toll-like receptor 2/6 (TLR2/6) signaling in tumor cells is regarded as one of the mechanisms of chronic inflammation but it can also mediate tumor cell immune escape and tumor progression. However, the expression of TLR2 and its biological function in the development and progression of hepatocarcinoma have not been investigated. This study aimed to determine the expression of TLRs 1–10 in the established human hepatocellular carcinoma cell line BLE-7402, to investigate the biological effect of TLR2 on cell growth and survival.

Methods

TLR expression in BLE-7402 cells was assayed by RT-PCR, real-time PCR and flow cytometry (FCM). To further investigate the function of TLR2 in hepatocarcinoma growth, BLE-7402 cells were transfected with recombinant plasmids expressing one of three forms of TLR2 siRNA (sh-TLR2 RNAi(A, B and C)). TLR2 knockdown was confirmed using RT-PCR, real-time PCR and fluorescence microscopy. Tumor cell proliferation was monitored by MTT assay and secreted cytokines in the supernatant of transfected cells were measured by bead-based FCM, the function of TLR2 siRNA was also investigated in vivo.

Results

The BLE-7402 cell line expressed TLRs 2 to 10 at both mRNA and protein levels. TLR2 was the most highly expressed TLR. While all the three siRNAs inhibited TLR2 mRNA and protein expression, sh-TLR2 RNAi(B) had the strongest knockdown effect. TLR2 knockdown with sh-TLR2 RNAi(B) reduced cell proliferation. Furthermore, secretion of IL-6 and IL-8 was also reduced. The result showed a drastic reduction in tumor volume in mice treated with sh-TLR2 RNAi(B).

Discussion

These results suggest that TLR2 knockdown inhibit proliferation of cultured hepatocarcinoma cells and decrease the secretion of cytokines. It is suggested that TLR2 silencing may worth further investigations for siRNA based gene therapy in treatment of hepatocarcinoma.