CAFET algorithm reveals Wnt/PCP signature in lung squamous cell carcinoma

We analyzed the gene expression patterns of 138 Non-Small Cell Lung Cancer (NSCLC) samples and developed a new algorithm called Coverage Analysis with Fisher's Exact Test (CAFET) to identify molecular pathways that are differentially activated in squamous cell carcinoma (SCC) and adenocarcinoma (AC) subtypes. Analysis of the lung cancer samples demonstrated hierarchical clustering according to the histological subtype and revealed a strong enrichment for the Wnt signaling pathway components in the cluster consisting predominantly of SCC samples. The specific gene expression pattern observed correlated with enhanced activation of the Wnt Planar Cell Polarity (PCP) pathway and inhibition of the canonical Wnt signaling branch. Further real time RT-PCR follow-up with additional primary tumor samples and lung cancer cell lines confirmed enrichment of Wnt/PCP pathway associated genes in the SCC subtype. Dysregulation of the canonical Wnt pathway, characterized by increased levels of β-catenin and epigenetic silencing of negative regulators, has been reported in adenocarcinoma of the lung. Our results suggest that SCC and AC utilize different branches of the Wnt pathway during oncogenesis.     

Identification of Logic Relationships between Genes and Subtypes of Non-Small Cell Lung Cancer

Non-small cell lung cancer (NSCLC) has two major subtypes: adenocarcinoma (AC) and squamous cell carcinoma (SCC). The diagnosis and treatment of NSCLC are hindered by the limited knowledge about the pathogenesis mechanisms of subtypes of NSCLC. It is necessary to research the molecular mechanisms related with AC and SCC. In this work, we improved the logic analysis algorithm to mine the sufficient and necessary conditions for the presence states (presence or absence) of phenotypes. We applied our method to AC and SCC specimens, and identified lower and higher logic relationships between genes and two subtypes of NSCLC. The discovered relationships were independent of specimens selected, and their significance was validated by statistic test. Compared with the two earlier methods (the non-negative matrix factorization method and the relevance analysis method), the current method outperformed these methods in the recall rate and classification accuracy on NSCLC and normal specimens. We obtained biomarkers. Among biomarkers, genes have been used to distinguish AC from SCC in practice, and other six genes were newly discovered biomarkers for distinguishing subtypes. Furthermore, NKX2-1 has been considered as a molecular target for the targeted therapy of AC, and other genes may be novel molecular targets. By gene ontology analysis, we found that two biological processes (‘epidermis development’ and ‘cell adhesion’) were closely related with the tumorigenesis of subtypes of NSCLC. More generally, the current method could be extended to other complex diseases for distinguishing subtypes and detecting the molecular targets for targeted therapy.     

Gene Expression Signature Differentiates Histology But Not Progression Status of Early-Stage NSCLC

Advances in molecular analyses based on high-throughput technologies can contribute to a more accurate classification of non–small cell lung cancer (NSCLC), as well as a better prediction of both the disease course and the efficacy of targeted therapies. Here we set out to analyze whether global gene expression profiling performed in a group of early-stage NSCLC patients can contribute to classifying tumor subtypes and predicting the disease prognosis. Gene expression profiling was performed with the use of the microarray technology in a training set of 108 NSCLC samples. Subsequently, the recorded findings were validated further in an independent cohort of 44 samples. We demonstrated that the specific gene patterns differed significantly between lung adenocarcinoma (AC) and squamous cell lung carcinoma (SCC) samples. Furthermore, we developed and validated a novel 53-gene signature distinguishing SCC from AC with 93% accuracy. Evaluation of the classifier performance in the validation set showed that our predictor classified the AC patients with 100% sensitivity and 88% specificity. We revealed that gene expression patterns observed in the early stages of NSCLC may help elucidate the histological distinctions of tumors through identification of different gene-mediated biological processes involved in the pathogenesis of histologically distinct tumors. However, we showed here that the gene expression profiles did not provide additional value in predicting the progression status of the early-stage NSCLC. Nevertheless, the gene expression signature analysis enabled us to perform a reliable subclassification of NSCLC tumors, and it can therefore become a useful diagnostic tool for a more accurate selection of patients for targeted therapies.     

Wnt5a is strongly expressed at the leading edge in non-melanoma skin cancer, forming active gradients, while canonical Wnt signalling is repressed

Wnt5a is one of the so-called non-canonical Wnt ligands which do not act through β-catenin. In normal development, Wnt5a is secreted and directs the migration of target cells along concentration gradients. The effect of Wnt5a on target cells is regulated by many factors, including the expression level of inhibitors and receptors. Dysregulated Wnt5a signalling facilitates invasion of multiple tumor types into adjacent tissue. However, the expression and distribution of Wnt5a in cutaneous squamous cell carcinoma (SCC) and basal cell carcinoma (BCC), as well as the effect of Wnt5a on keratinocyte migration has not been studied in detail to date. We here report that Wnt5a is upregulated in SCC and BCC and localised to the leading edge of tumors, as well as tumor-associated fibroblasts. The Wnt5a-triggered bundling of its receptor Fzd3 provides evidence of Wnt5a concentration gradients projecting into the tumor. In vitro migration assays show that Wnt5a concentration gradients determine its effect on keratinoctye migration: While chemotactic migration is inhibited by Wnt5a present in homogenous concentrations, it is enhanced in the presence of a Wnt5a gradient. Expression profiling of the Wnt pathway shows that the upregulation of Wnt5a in SCC is coupled to repression of canonical Wnt signalling. This is confirmed by immunohistochemistry showing lack of nuclear β-catenin, as well as absent accumulation of Axin2. Since both types of Wnt signalling act mutually antogonistically at multiple levels, the concurrent repression of canonical Wnt signalling suggests hyper-active Wnt5a signal transduction. Significantly, this combination of gene dysregulation is not observed in the benign hyperproliferative inflammatory skin disease psoriasis. Collectively, our data strongly suggest that Wnt5a signalling contributes to tissue invasion by non-melanoma skin cancer.     

Macrophages Mediate a Switch between Canonical and Non-Canonical Wnt Pathways in Canine Mammary Tumors

Objective

According to the current hypothesis, tumor-associated macrophages (TAMs) are “corrupted” by cancer cells and subsequently facilitate, rather than inhibit, tumor metastasis. Because the molecular mechanisms of cancer cell–TAM interactions are complicated and controversial we aimed to better define this phenomenon.

Methods and Results

Using microRNA microarrays, Real-time qPCR and Western blot we showed that co-culture of canine mammary tumor cells with TAMs or treatment with macrophage-conditioned medium inhibited the canonical Wnt pathway and activated the non-canonical Wnt pathway in tumor cells. We also showed that co-culture of TAMs with tumor cells increased expression of canonical Wnt inhibitors in TAMs. Subsequently, we demonstrated macrophage-induced invasive growth patterns and epithelial–mesenchymal transition of tumor cells. Validation of these results in canine mammary carcinoma tissues (n = 50) and xenograft tumors indicated the activation of non-canonical and canonical Wnt pathways in metastatic tumors and non-metastatic malignancies, respectively. Activation of non-canonical Wnt pathway correlated with number of TAMs.

Conclusions

We demonstrated that TAMs mediate a “switch” between canonical and non-canonical Wnt signaling pathways in canine mammary tumors, leading to increased tumor invasion and metastasis.Interestingly, similar changes in neoplastic cells were observed in the presence of macrophage-conditioned medium or live macrophages. These observations indicate that rather than being “corrupted” by cancer cells, TAMs constitutively secrete canonical Wnt inhibitors that decrease tumor proliferation and development, but as a side effect, they induce the non-canonical Wnt pathway, which leads to tumor metastasis.These data challenge the conventional understanding of TAM–cancer cell interactions.     

Epigenetic loss of putative tumor suppressor SFRP3 correlates with poor prognosis of lung adenocarcinoma patients

Secreted frizzled related protein 3 (SFRP3) contains a cysteine-rich domain (CRD) that shares homology with Frizzled CRD and regulates WNT signaling. Independent studies showed epigenetic silencing of SFRP3 in melanoma and hepatocellular carcinoma. Moreover, a tumor suppressive function of SFRP3 was shown in androgen-independent prostate and gastric cancer cells. The current study is the first to investigate SFRP3 expression and its potential clinical impact on non-small cell lung carcinoma (NSCLC). WNT signaling components present on NSCLC subtypes were preliminary elucidated by expression data of The Cancer Genome Atlas (TCGA). We identified a distinct expression signature of relevant WNT signaling components that differ between adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). Of interest, canonical WNT signaling is predominant in LUAD samples and non-canonical WNT signaling is predominant in LUSC. In line, high SFRP3 expression resulted in beneficial clinical outcome for LUAD but not for LUSC patients. Furthermore, SFRP3 mRNA expression was significantly decreased in NSCLC tissue compared to normal lung samples. TCGA data verified the reduction of SFRP3 in LUAD and LUSC patients. Moreover, DNA hypermethylation of SFRP3 was evaluated in the TCGA methylation dataset resulting in epigenetic inactivation of SFRP3 expression in LUAD, but not in LUSC, and was validated by pyrosequencing of our NSCLC tissue cohort and in vitro demethylation experiments. Immunohistochemistry confirmed SFRP3 protein downregulation in primary NSCLC and indicated abundant expression in normal lung tissue. Two adenocarcinoma gain-of-function models were used to analyze the functional impact of SFRP3 on cell proliferation and regulation of CyclinD1 expression in vitro. Our results indicate that SFRP3 acts as a novel putative tumor suppressor gene in adenocarcinoma of the lung possibly regulating canonical WNT signaling.     

Phasic modulation of Wnt signaling enhances cardiac differentiation in human pluripotent stem cells by recapitulating developmental ontogeny

Cardiomyocytes (CMs) derived from human pluripotent stem cells (hPSCs) offer immense value in studying cardiovascular regenerative medicine. However, intrinsic biases and differential responsiveness of hPSCs towards cardiac differentiation pose significant technical and logistic hurdles that hamper human cardiomyocyte studies. Tandem modulation of canonical and non-canonical Wnt signaling pathways may play a crucial role in cardiac development that can efficiently generate cardiomyocytes from pluripotent stem cells. Our Wnt signaling expression profiles revealed that phasic modulation of canonical/non-canonical axis enabled orderly recapitulation of cardiac developmental ontogeny. Moreover, evaluation of 8 hPSC lines showed marked commitment towards cardiac-mesoderm during the early phase of differentiation, with elevated levels of canonical Wnts (Wnt3 and 3a) and Mesp1. Whereas continued activation of canonical Wnts was counterproductive, its discrete inhibition during the later phase of cardiac differentiation was accompanied by significant up-regulation of non-canonical Wnt expression (Wnt5a and 11) and enhanced Nkx2.5⁺ (up to 98%) populations. These Nkx2.5⁺ populations transited to contracting cardiac troponin T-positive CMs with up to 80% efficiency. Our results suggest that timely modulation of Wnt pathways would transcend intrinsic differentiation biases of hPSCs to consistently generate functional CMs that could facilitate their scalable production for meaningful clinical translation towards personalized regenerative medicine.     

Activation of non-canonical Wnt/JNK pathway by Wnt3a is associated with differentiation fate determination of human bone marrow stromal (mesenchymal) stem cells

The canonical Wnt signaling pathway can determine human bone marrow stromal (mesenchymal) stem cell (hMSC) differentiation fate into osteoblast or adipocyte lineages. However, its downstream targets in MSC are not well characterized. Thus, using DNA microarrays, we compared global gene expression patterns induced by Wnt3a treatment in two hMSC lines: hMSC-LRP5^T253 and hMSC-LRP5^T244 cells carrying known mutations of Wnt co-receptor LRP5 (T253I or T244M) that either enhances or represses canonical Wnt signaling, respectively. Wnt3a treatment of hMSC activated not only canonical Wnt signaling, but also the non-canonical Wnt/JNK pathway through upregulation of several non-canonical Wnt components e.g. naked cuticle 1 homolog (NKD1) and WNT11. Activation of the non-canonical Wnt/JNK pathway by anisomycin enhanced osteoblast differentiation whereas its inhibition by SP600125 enhanced adipocyte differentiation of hMSC. In conclusion, canonical and non-canonical Wnt signaling cooperate in determining MSC differentiation fate.     

Both Canonical and Non-Canonical Wnt Signaling Independently Promote Stem Cell Growth in Mammospheres

The characterization of mammary stem cells, and signals that regulate their behavior, is of central importance in understanding developmental changes in the mammary gland and possibly for targeting stem-like cells in breast cancer. The canonical Wnt/β-catenin pathway is a signaling mechanism associated with maintenance of self-renewing stem cells in many tissues, including mammary epithelium, and can be oncogenic when deregulated. Wnt1 and Wnt3a are examples of ligands that activate the canonical pathway. Other Wnt ligands, such as Wnt5a, typically signal via non-canonical, β-catenin-independent, pathways that in some cases can antagonize canonical signaling. Since the role of non-canonical Wnt signaling in stem cell regulation is not well characterized, we set out to investigate this using mammosphere formation assays that reflect and quantify stem cell properties. Ex vivo mammosphere cultures were established from both wild-type and Wnt1 transgenic mice and were analyzed in response to manipulation of both canonical and non-canonical Wnt signaling. An increased level of mammosphere formation was observed in cultures derived from MMTV-Wnt1 versus wild-type animals, and this was blocked by treatment with Dkk1, a selective inhibitor of canonical Wnt signaling. Consistent with this, we found that a single dose of recombinant Wnt3a was sufficient to increase mammosphere formation in wild-type cultures. Surprisingly, we found that Wnt5a also increased mammosphere formation in these assays. We confirmed that this was not caused by an increase in canonical Wnt/β-catenin signaling but was instead mediated by non-canonical Wnt signals requiring the receptor tyrosine kinase Ror2 and activity of the Jun N-terminal kinase, JNK. We conclude that both canonical and non-canonical Wnt signals have positive effects promoting stem cell activity in mammosphere assays and that they do so via independent signaling mechanisms.     

Non-Canonical Wnt Predominates in Activated Rat Hepatic Stellate Cells,Influencing HSC Survival and Paracrine Stimulation of Kupffer Cells

The Wnt system is highly complex and is comprised of canonical and non-canonical pathways leading to the activation of gene expression. Our aim was to examine changes in the expression of Wnt ligands and regulators during hepatic stellate cell (HSC) transdifferentiation and assess the relative contributions of the canonical and non-canonical Wnt pathways in fibrogenic activated HSC. The expression profile of Wnt ligands and regulators in HSC was not supportive for a major role for β-catenin-dependent canonical Wnt signalling, this verified by inability to induce Topflash reporter activity in HSC even when expressing a constitutive active β-catenin. We detected expression of Wnt5a in activated HSC which can signal via non-canonical mechanisms and showed evidence for non-canonical signalling in these cells involving phosphorylation of Dvl2 and pJNK. Stimulation of HSC or Kupffer cells with Wnt5a regulated HSC apoptosis and expression of TGF-β1 and MCP1 respectively. We were unable to confirm a role for β-catenin-dependent canonical Wnt in HSC and instead propose autocrine and paracrine functions for Wnts expressed by activated HSC via non-canonical pathways. The data warrant detailed investigation of Wnt5a in liver fibrosis.     

KCTD11 inhibits progression of lung cancer by binding to β-catenin to regulate the activity of the Wnt and Hippo pathways

KCTD11 has been reported to be a potential tumour suppressor in several tumour types. However, the expression of KCTD11 and its role has not been reported in human non-small cell lung cancer (NSCLC). Whether its potential molecular mechanism is related to its BTB domain is also unknown. The expression of KCTD11 in 139 NSCLC tissue samples was detected by immunohistochemistry, and its correlation with clinicopathological factors was analysed. The effect of KCTD11 on the biological behaviour of lung cancer cells was verified in vitro and in vivo. Its effect on the epithelial-mesenchymal transition(EMT)process and the Wnt/β-catenin and Hippo/YAP pathways were observed by Western blot, dual-luciferase assay, RT-qPCR, immunofluorescence and immunoprecipitation. KCTD11 is under-expressed in lung cancer tissues and cells and was negatively correlated with the degree of differentiation, tumour-node-metastasis (TNM) stage and lymph node metastasis. Low KCTD11 expression was associated with poor prognosis. KCTD11 overexpression inhibited the proliferation and migration of lung cancer cells. Further studies indicated that KCTD11 inhibited the Wnt pathway, activated the Hippo pathway and inhibited EMT processes by inhibiting the nuclear translocation of β-catenin and YAP. KCTD11 lost its stimulatory effect on the Hippo pathway after knock down of β-catenin. These findings confirm that KCTD11 inhibits β-catenin and YAP nuclear translocation as well as the malignant phenotype of lung cancer cells by interacting with β-catenin. This provides an important experimental basis for the interaction between KCTD11, β-catenin and YAP, further revealing the link between the Wnt and Hippo pathways.     

基于蛋白互作网络识别非小细胞肺癌相关基因功能模块

本研究对非小细胞肺癌(non-small cell lung carcinoma,NSCLC)基因表达数据进行差异表达分析,并与蛋白质相互作用网络(PPIN)数据进行整合,进一步利用Heinz搜索算法识别NSCLC相关的基因功能模块,并对模块中的基因进行功能(GO term)和通路(KEGG)富集分析,旨在探究肺癌发病分子机制。蛋白互作网络分析得到一个包含96个基因和117个相互作用的功能模块,以及8个对NSCLC的发生和发展起到关键作用候选基因标志物。富集分析结果表明,这些基因主要富集于基因转录催化及染色质调控等生物学过程,并在基础转录因子、黏着连接、细胞周期、Wnt信号通路及HTLV-Ⅰ感染等生物学通路中发挥重要作用。本研究对非小细胞肺癌相关的基因和生物学通路进行预测,可用于肺癌的早期诊断和早期治疗,以降低肺癌死亡率。     

Distinguishing Lung Adenocarcinoma from Lung Squamous Cell Carcinoma by Two Hypomethylated and Three Hypermethylated Genes: A Meta-Analysis

Significant differences in the aberrant methylation of genes exist among various histological types of non-small cell lung cancer (NSCLC), which includes adenocarcinoma (AC) and squamous cell carcinoma (SCC). Different chemotherapeutic regimens should be administered to the two NSCLC subtypes due to their unique genetic and epigenetic profiles. The purpose of this meta-analysis was to generate a list of differentially methylated genes between AC and SCC. Our meta-analysis encompassed 151 studies on 108 genes among 12946 AC and 10243 SCC patients. Our results showed two hypomethylated genes (CDKN2A and MGMT) and three hypermethylated genes (CDH13, RUNX3 and APC) in ACs compared with SCCs. In addition, our results showed that the pooled specificity and sensitivity values of CDH13 and APC were higher than those of CDKN2A, MGMT and RUNX3. Our findings might provide an alternative method to distinguish between the two NSCLC subtypes.     

Zebrafish Naked1 and Naked2 antagonize both canonical and non-canonical Wnt signaling

Wnt signaling controls a wide range of developmental processes and its aberrant regulation can lead to disease. To better understand the regulation of this pathway, we identified zebrafish homologues of Naked Cuticle (Nkd), Nkd1 and Nkd2, which have previously been shown to inhibit canonical Wnt/beta-catenin signaling. Zebrafish nkd1 expression increases substantially after the mid-blastula transition in a pattern mirroring that of activated canonical Wnt/beta-catenin signaling, being expressed in both the ventrolateral blastoderm margin and also in the axial mesendoderm. In contrast, zebrafish nkd2 is maternally and ubiquitously expressed. Overexpression of Nkd1 or Nkd2a suppressed canonical Wnt/beta-catenin signaling at multiple stages of early zebrafish development and also exacerbated the cyclopia and axial mesendoderm convergence and extension (C&E) defect in the non-canonical Wnt/PCP mutant silberblick (slb/wnt11). Thus, Nkds are sufficient to antagonize both canonical and non-canonical Wnt signaling. Reducing Nkd function using antisense morpholino oligonucleotides resulted in increased expression of canonical Wnt/beta-catenin target genes. Finally, reducing Nkd1 function in slb mutants suppressed the axial mesendoderm C&E defect. These data indicate that zebrafish Nkd1 and Nkd2 function to limit both canonical and non-canonical Wnt signaling.     

波形蛋白在非小细胞肺癌组织中的表达及其临床意义

目的:探究波形蛋白在非小细胞肺癌(NSCLC)组织中的表达及其与肺癌浸润转移的相关性。方法:收集2012年6月-2014年6月我院手术切除的NSCLC癌组织标本150例及癌旁正常组织(距肿瘤5 cm)79例,提取两组的RNA,采用实时荧光定量聚合酶链反应(RT-PCR)检测波形蛋白m RNA表达水平,免疫组化法检测波形蛋白的蛋白表达,分析波形蛋白表达水平与淋巴结转移、TNM分期的相关性。结果:波形蛋白m RNA在NSCLC癌组织中的表达明显高于癌旁正常组织(P0.05)。NSCLC癌组织中波形蛋白m RNA表达水平的上调与淋巴结转移及TNM分期(P0.05)相关。结论:波形蛋白在NSCLC患者中表达异常升高,与NSCLC的发生和浸润转移密切相关。     

Wnt5a and Wnt11 interact in a maternal Dkk1-regulated fashion to activate both canonical and non-canonical signaling in Xenopus axis formation

Wnt signaling in development and adult tissue homeostasis requires tight regulation to prevent patterning abnormalities and tumor formation. Here, we show that the maternal Wnt antagonist Dkk1 downregulates both the canonical and non-canonical signaling that are required for the correct establishment of the axes of the Xenopus embryo. We find that the target Wnts of Dkk activity are maternal Wnt5a and Wnt11, and that both Wnts are essential for canonical and non-canonical signaling. We determine that Wnt5a and Wnt11 form a previously unrecognized complex. This work suggests a new aspect of Wnt signaling: two Wnts acting in a complex together to regulate embryonic patterning.