Ovarian Cancer Cell Heparan Sulfate 6-O-Sulfotransferases Regulate an Angiogenic Program Induced by Heparin-binding Epidermal Growth Factor (EGF)-like Growth Factor/EGF Receptor Signaling

Heparan sulfate (HS) is a component of cell surface and extracellular matrix proteoglycans that regulates numerous signaling pathways by binding and activating multiple growth factors and chemokines. The amount and pattern of HS sulfation are key determinants for the assembly of the trimolecular, HS-growth factor-receptor, signaling complex. Here we demonstrate that HS 6-O-sulfotransferases 1 and 2 (HS6ST-1 and HS6ST-2), which perform sulfation at 6-O position in glucosamine in HS, impact ovarian cancer angiogenesis through the HS-dependent HB-EGF/EGFR axis that subsequently modulates the expression of multiple angiogenic cytokines. Down-regulation of HS6ST-1 or HS6ST-2 in human ovarian cancer cell lines results in 30–50% reduction in glucosamine 6-O-sulfate levels in HS, impairing HB-EGF-dependent EGFR signaling and diminishing FGF2, IL-6, and IL-8 mRNA and protein levels in cancer cells. These cancer cell-related changes reduce endothelial cell signaling and tubule formation in vitro. In vivo, the development of subcutaneous tumor nodules with reduced 6-O-sulfation is significantly delayed at the initial stages of tumor establishment with further reduction in angiogenesis occurring throughout tumor growth. Our results show that in addition to the critical role that 6-O-sulfate moieties play in angiogenic cytokine activation, HS 6-O-sulfation level, determined by the expression of HS6ST isoforms in ovarian cancer cells, is a major regulator of angiogenic program in ovarian cancer cells impacting HB-EGF signaling and subsequent expression of angiogenic cytokines by cancer cells.     

Expression of Duffy antigen receptor for chemokines (DARC) is down-regulated in colorectal cancer

Abstract

Duffy antigen receptor for chemokines (DARC) is a silent chemokine receptor which selectively binds angiogenic chemokines without inducing conventional signaling responses. DARC has been reported to inhibit the development of multiple cancers through clearance of angiogenic chemokines. However, its role in colorectal cancer (CRC) remains unclear. We investigated the expression of DARC in CRC and explored correlation of DARC expression with clinical pathological features and microvessel density (MVD). The protein expression levels of DARC were detected by immunohistochemistry in 90 CRC and 64 paired unaffected tissues. The mRNA levels of DARC were detected by quantitative real-time PCR in 15 CRC and paired unaffected tissues. MVD in CRC was also assessed by immunohistochemistry of CD34. We found that the mRNA and protein expression levels of DARC were significantly lower in CRC than in the unaffected tissues (p?<?0.05). The DARC protein expression levels were positively correlated with DARC mRNA expression levels in both CRC (p?<?0.001) and unaffected tissues (p?<?0.001). We also found that DARC expression was significantly correlated with tumor differentiation (p?<?0.001), lymph node metastasis (p?<?0.01) and TNM stage (p?<?0.05). Moreover, we observed a strong negative relationship between DARC expression and MVD in CRC (p?<?0.001). We showed that DARC expression is down-regulated in CRC and associated with clinical pathological features and MVD of CRC. DARC might be involved in tumorigenesis, progression, angiogenesis, and metastasis of CRC.     

Elevation of circulating interleukin-8 is related to lymph node and distant metastases in esophageal squamous cell carcinomas--implication for clinical evaluation of cancer patient

The presence of lymph node metastasis (LNM) is an important factor in clinical evaluation of esophageal cancer patients. Biological markers able to support detection of metastatic lymph nodes are sought after. Interleukin-8 (IL-8) is overexpressed by many cancers and involved in cancer dissemination. We investigated the relationship between circulating IL-8 and clinicopathological features of esophageal squamous cell carcinoma (ESCC), and evaluated the diagnostic potential of IL-8, with reference to the key angiogenic and lymphangiogenic factors: vascular endothelial growth factors A and C (VEGF-A and VEGF-C). We found elevated IL-8 levels in ESCC patients, correlated with tumor size and cancer dissemination, especially LNM. Circulating IL-8 correlated with lymphangiogenic VEGF-C rather then angiogenic VEGF-A. The association weakened in metastatic cancers, suggesting divergent mechanism of IL-8 involvement in the dissemination process. The cytokine levels correlated with platelets and neutrophils, pointing at these cells as possible sources of circulating IL-8. We demonstrated IL-8 that positively correlated with inflammation status of ESCC patients. Circulating IL-8 was a better indicator of ESCC dissemination than VEGF-A or VEGF-C. Yet, the detection rates were not satisfactory enough to allow for the recommendation of IL-8 determination as an adjunct to the clinical evaluation of lymph node involvement in ESCC patients.     

Modulation of endothelial cell proliferation and capillary network formation by the ox-LDL component: 1-palmitoyl-2-archidonoyl-sn-glycero-3-phosphocholine (ox-PAPC)

Atherosclerotic plaque formation is often associated with pathological angiogenesis. Modified phospholipids, including oxidized lipoproteins such as LDL, are found to induce adhesion of the monocytes to the endothelial cells and to stimulate their chemotaxis. Effects of oxidized 1-palmitoyl-2-archidonoyl-sn-glycero-3-phosphocholine (ox-PAPC) mimic actions of minimally modified LDL in vivo. Interleukin-8 (IL-8) and interleukin-15 (IL-15) are known to induce both inflammation and angiogenesis. The goal of our study was to analyze a potential synergism between ox-PAPC and IL-15 in the in vitro model of angiogenesis carried out in the human endothelial cells (HUVECs). Increasing IL-15 concentrations led to formation of the tube-like structures in the matrigel 3D-model of angiogenesis (P < 0.05), in contrast to ox-PAPC that inhibited this process. HUVECs incubation with ox-PAPC led to reduced IL-15 gene basal expression (P = 0.033) along with parallel increase, however statistically insignificant, of basal gene expression of IL-8 (P = 0.086). Our findings point to the ox-PAPC opposite effects on the IL-8- and IL-15-mediated angiogenic responses that contribute to pathological angiogenesis induced by ox-LDL.     

REC8 enhances stemness and promotes metastasis of colorectal cancer through BTK/Akt/β-catenin signaling pathway

Cancer/testis antigens (CTAs) are often aberrantly expressed in cancer stem cells (CSCs) which are responsible for tumor metastasis. Rec8 meiotic recombination protein (REC8), a member of CTAs, shares distinct roles in various cancers, while its contribution to CSCs and colorectal cancer (CRC) remains unclear. We found that overexpression of REC8 facilitated the migration and invasion of CRC cells (DLD-1 and SW480 cells) in vitro and promoted the liver metastasis of CRC in vivo. Moreover, REC8 is highly expressed in CRC stem-like cells and is required for the maintenance of CSC stemness. Mechanistic studies suggested that REC8 mediated through the activation of Bruton tyrosine kinase (BTK). Inhibition of BTK by ibrutinib not only suppressed the migration and invasion-promoting ability, but also declined the increased expression of p-BTK, p-Akt, β-catenin, and CSC markers upon REC8 overexpression. Importantly, high expression of REC8 in cancerous tissues was related to advanced clinical stage and lymph node metastasis of 62 CRC patients, and REC8 was enriched in the cancerous cells positive for CSC markers. Collectively, our results indicate that REC8 promotes CRC metastasis by increasing cell stemness through BTK/Akt/β-catenin pathway.     

CCNB2在结直肠癌中的表达及临床意义

目的:探讨细胞周期蛋白B2(Cyclin B2,CCNB2)在结直肠癌组织中的表达及其临床意义。方法:选择45对结直肠癌组织及癌旁正常结直肠组织样本,分别采用实时定量PCR(qRT-PCR)方法和免疫组织化学技术检测CCNB2的mRNA和蛋白表达,并进一步分析CCNB2的表达与结直肠癌临床病理特征之间的关系。结果:结直肠癌组织中CCNB2 mRNA的表达显著高于癌旁正常结直肠组织,差异有统计学意义(P0.001),且CCNB2的mRNA表达与结直肠癌的肿瘤大小、浸润深度及TNM分期显著相关(P0.05),与年龄、性别、肿瘤位置、分化程度、脉管神经浸润、淋巴结转移和远处转移均无关(P0.05)。45例结直肠癌标本中39例表达(+~+++),6例表达(-)。CCNB2蛋白主要表达于结直肠癌细胞质中,少量见于细胞核。结直肠癌组织中CCNB2蛋白的阳性表达率为86.7%,显著高于癌旁正常结直肠组织,并与患者的性别、年龄、分化程度和肿瘤转移均无显著相关性(P0.05),但与肿瘤分期、浸润程度均显著相关(P0.05)。结论:CCNB2在结直肠癌中呈异常高表达,且与结直肠癌的发生发展相关,有望作为结直肠癌的诊断和预后预测参考指标。     

Regulation of neurokine receptor signaling and trafficking

Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) are neurally active cytokines, or neurokines. LIF signals through a receptor consisting of gp130 and the low affinity LIF receptor (LIFR), while the CNTF receptor consists of gp130, LIFR, and the low affinity CNTF receptor (CNTFR). Ser1044 of the LIFR is phosphorylated by Erk1/2 MAP kinase. Stimulation of neural cells with growth factors which strongly activate Erk1/2 decreases LIF-mediated signal transduction due to increased degradation of the LIFR as a consequence of Erk1/2-dependent phosphorylation of the receptor at Ser1044.     

Silencing NPAS2 promotes cell growth and invasion in DLD-1 cells and correlated with poor prognosis of colorectal cancer

Emerging evidences show that circadian rhythm disorder is an important factor of tumor initiation and development. Neuronal PAS domain protein2 (NPAS2), which is the largest circadian gene, has been proved to be a novel prognostic biomarker in breast cancer and non-Hodgkin’s lymphoma. However, the potential functions of NPAS2 in colorectal cancer are still unknown. In our present study, we detected the mRNA expressions of NPAS2 in 108 CRC patients by RT-PCR, and found that NPAS2 expression was significantly down-regulated in tumor tissues than that in NATs. Clinicopathologic analysis revealed that low expression of NPAS2 was associated with the tumor size, TNM stage and tumor distance metastasis in colorectal cancer (p < 0.05). Furthermore, we effectively down-regulated NPAS2 mRNA expression by transfecting RNA interfere fragments into DLD-1 cells, and our results in vitro demonstrated that silencing NPAS2 expression could promote cell proliferation, cell invasion and increase the wound healing ability (p < 0.05). However, down-regulating NPAS2 expression did not influence the apoptotic rate in DLD-1 cells (p > 0.05). In conclusion, our study suggested that NPAS2, functioned as a potential tumor suppressor gene, could serve as a promising target and potential prognostic indicator for colorectal cancer.     

Overexpression of integrin-linked kinase (ILK) is associated with tumor progression and an unfavorable prognosis in patients with colorectal cancer

Integrin-linked kinase (ILK), an intracellular serine-threonine kinase, has been reported to be overexpressed in multiple types of human malignancies, including colorectal cancer (CRC). The prognostic value of ILK in CRC, however, remains unknown. In the present study, expression of ILK in 25 paired primary CRC samples and adjacent noncancerous tissues were quantified using real-time PCR and Western blotting. ILK protein expression was analyzed in 102 archived, paraffin-embedded CRC samples using immunohistochemistry. The correlation between ILK expression and clinicopathological factors was evaluated by the χ² test. Patients’ overall survival was analyzed by Kaplan–Meier method. We found that both ILK mRNA and protein expression levels were significantly up-regulated in primary CRC samples compared with their corresponding normal tissues. Immunohistochemical analysis revealed relative high expression of ILK in 43 of 102 (42.2 %) primary CRC samples. Statistical analysis showed a significant correlation of ILK expression with tumor differentiation, lymph node metastasis, tumor invasion, and tumor-node-metastasis stage. Patients with tumors displaying high-level ILK expression showed significantly shorter overall survival (P = 0.028, log-rank test). More importantly, multivariate analysis indicated that high ILK protein expression was an independent prognostic factor for CRC patients (P = 0.026). Taken together, our data suggest that ILK overexpression is associated with tumor progression and a poor prognosis in CRC patients and may represent a novel potential prognostic marker for patients with CRC.     

Serum cytokine profile as a potential prognostic tool in colorectal cancer patients – one center study

AimComparison of 14 cytokines levels between a control group and prospectively enrolled CRC patients to confirm their significance in CRC development. We tested if a model based on 14 cytokines levels could predict prognosis in Caucasian CRC patients treated with 5-FU based chemotherapy.BackgroundNovel prognostic tools in colorectal cancer (CRC) are necessary to optimize treatment, reduce toxicity and chemotherapy (CHT) costs.Materials and MethodsWe assessed prognostic significance of 14 cytokines: IL-1 beta, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL12p70, IL-13, IL-17A in 75 prospectively enrolled CRC patients before initiation of palliative or adjuvant CHT and in 22 control subjects. Readings were taken using the Bio-Plex 200 System. Response to treatment was assessed after 6 months from initiation of CHT. The treated group was divided depending on the response into a progressors (death, progression of disease) and non-progressors group (stable disease, partial response, complete response).ResultsWe found that increased concentration of IL-8 was a negative prognostic factor in the whole group and palliative subgroup, whereas increased level of IL-10, IL-7, and IL-12p70 was a negative predictor in the adjuvant group CHT.ConclusionsWe proposed a statistical model based on circulating cytokine levels, showing a good prognostic value in prediction of the response to CHT (AUC = 0.956). The model, including combined IL-2, IL-8, IL-10 and IL-13 levels, established in the whole treated group, should be validated in larger trials.     

Plectin promotes migration and invasion of cancer cells and is a novel prognostic marker for head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is usually found at a late stage and distant metastasis occurs at high frequency; therefore, novel prognostic markers are needed. This study was aimed to identify novel tumor markers in HNSCC. We identified 65 proteins which were significantly increased or decreased in the tumors by 2D-DIGE using 12 HNSCC and adjacent non-cancer tissues. Western blotting and immunohistochemical analysis confirmed that the expression of plectin was significantly increased in most cancer tissues as compared with non-cancer tissues. Strikingly, the suppression of endogenous plectin using siRNA inhibited the proliferation, migration and invasion of HNSCC cells and down-regulated Erk 1/2 kinase. Furthermore, immunohistochemistry using paraffin-embedded tissues from 62 patients showed not only that the frequency of recurrence was correlated with the plectin expression but that the prognosis of patients with a high plectin was extremely poor. Moreover, the survival rate of patients with a high plectin was significantly lower than that of patients with low E-cadherin levels, which is known to correlate with the poor prognosis of HNSCC. Our findings suggest that plectin promotes the migration and invasion of HNSCC cells through activation of Erk 1/2 kinase and is a potential prognostic biomarker of HNSCC.     

Brain-Derived Neurotrophic Factor (BDNF)-Induced Tropomyosin-Related Kinase B (Trk B) Signaling Is a Potential Therapeutic Target for Peritoneal Carcinomatosis Arising from Colorectal Cancer

Tropomyosin-related receptor kinase B (TrkB) signaling, stimulated by brain-derived neurotrophic factor (BDNF) ligand, promotes tumor progression, and is related to the poor prognosis of various malignancies. We sought to examine the clinical relevance of BDNF/TrkB expression in colorectal cancer (CRC) tissues, its prognostic value for CRC patients, and its therapeutic potential in vitro and in vivo. Two hundred and twenty-three CRC patient specimens were used to determine both BDNF and TrkB mRNA levels. The expression of these proteins in their primary and metastatic tumors was investigated by immunohistochemistry. CRC cell lines and recombinant BDNF and K252a (a selective pharmacological pan-Trk inhibitor) were used for in vitro cell viability, migration, invasion, anoikis resistance and in vivo peritoneal metastasis assays. Tissue BDNF mRNA was associated with liver and peritoneal metastasis. Tissue TrkB mRNA was also associated with lymph node metastasis. The co-expression of BDNF and TrkB was associated with liver and peritoneal metastasis. Patients with higher BDNF, TrkB, and co-expression of BDNF and TrkB had a significantly poor prognosis. BDNF increased tumor cell viability, migration, invasion and inhibited anoikis in the TrkB-expressing CRC cell lines. These effects were suppressed by K252a. In mice injected with DLD1 co-expressing BDNF and TrkB, and subsequently treated with K252a, peritoneal metastatic nodules was found to be reduced, as compared with control mice. BDNF/TrkB signaling may thus be a potential target for treating peritoneal carcinomatosis arising from colorectal cancer.     

Autophagy triggered by magnolol derivative negatively regulates angiogenesis

Angiogenesis has a key role in the tumor progression and metastasis; targeting endothelial cell proliferation has emerged as a promising therapeutic strategy for the prevention of cancer. Previous studies have revealed a complex association between the process of angiogenesis and autophagy and its outcome on tumorigenesis. Autophagy, also known as type-II cell death, has been identified as an alternative way of cell killing in apoptotic-resistant cancer cells. However, its involvement in chemoresistance and tumor promotion is also well known. In this study, we used a derivate of natural product magnolol (Ery5), a potent autophagy inducer, to study the association between the autophagy and angiogenesis in both in vitro and in vivo model system. We found that the robust autophagy triggered by Ery5, inhibited angiogenesis and caused cell death independent of the apoptosis in human umbilical cord vein endothelial cells and PC-3 cells. Ery5 induced autophagy effectively inhibited cell proliferation, migration, invasion and tube formation. We further demonstrated that Ery5-mediated autophagy and subsequent inhibition of angiogenesis was reversed when autophagy was inhibited through 3-methyl adenine and knocking down of key autophagy proteins ATG7 and microtubule-associated protein light chain 3. While evaluating the negative regulation of autophagy on angiogenesis, it was interesting to find that angiogenic environment produced by the treatment of VEGF and CoCl2 remarkably downregulated the autophagy and autophagic cell death induced by Ery5. These studies, while disclosing the vital role of autophagy in the regulation of angiogenesis, also suggest that the potent modulators of autophagy can lead to the development of effective therapeutics in apoptosis-resistant cancer.     

Overexpression of PTP1B in human colorectal cancer and its association with tumor progression and prognosis

Protein tyrosine phosphatase 1B (PTP1B) is a non-transmembrane protein tyrosine phosphatase that has been implicated in cancer pathogenesis. However, the expression level and the role of PTP1B in the development and prognosis of colorectal cancer (CRC) remain unclear. In this study, the expression of PTP1B in CRC tissues and matched noncancerous tissues were detected by using immunohistochemistry, real-time PCR and Western blotting. The correlations between PTP1B expression level and clinicopathologic characteristics and patient survival were analyzed. We found that PTP1B expression was significantly higher in CRC tissues compared with matched non-tumour tissues. Statistical analysis showed that the PTP1B expression was correlated with tumor differentiation, tumor invasion, lymph node metastasis, and TNM stage. Patients with higher expressions of PTP1B had the lower survival (P = 0.012). Taken together, our results suggest that PTP1B expression might play a critical role in the progression of CRC and may serve as a valuable prognostic biomarker for CRC.     

Neuroendocrine differentiation contributes to radioresistance development and metastatic potential increase in non-small cell lung cancer

Radiation treatment induces neuroendocrine differentiation (NED) in non-small cell lung cancer (NSCLC) A549 and H157 cells, so higher NE-like features in radioresistant A549 (A549R26-1) and H157 (H157R24-1) cells are observed than in parental cells. We detected higher NED marker expressions in A549R26-1 cell-derived tumors than in A549 cell-derived tumors. In mechanism studies, we found that NED induction in A549R26-1 and H157R24-1 cells was accompanied by increased intracellular cAMP and IL-6 levels. Treatment of radioresistant lung cancer cells with the inhibitor (SQ22536) of adenylate cyclase (AC) which is the enzyme responsible for the cAMP production, or the neutralizing antibody (Ab) of IL-6, resulted in decreased NE-like features in radioresistant lung cancer cells. In addition, we found MEK/Erk is the signaling pathway that triggers the cAMP- and IL-6-mediated NED induction in radioresistant lung cancer cells. Also, we found that MEK/Erk signaling pathway inhibition decreased NED in radioresistant cells. Radioresistant lung cancer cells exhibiting high NE-like features also showed higher radioresistance and higher metastatic potential than parental cells. When we inhibited cAMP-, or IL-6-mediated pathways, or the downstream MEK/Erk signaling pathway, radiosensitivity of radioresistant lung cancer cells was significantly increased and their metastatic potential was significantly reduced. In in vivo mouse studies, reducing NED by treating mice with the MEK/Erk inhibitor increased radiosensitivity. Immunohistochemical staining of tumor tissues lowered expressions of the NED/epithelial-mesenchymal transition (EMT)/metastatic markers when mice were treated with the MEK/Erk inhibitor.