Arsenic acts as a human carcinogen and contributes to skin cancer via mechanisms that remain largely unknown. Recent evidence implicates the perturbation of Wnt, Shh and BMP signals as a potential mechanism. We initiated studies to examine gene expression changes in these signaling pathways. Meanwhile, the antagonistic effect of retinoic acid was explored. In this study, HaCaT and NHEK cells were treated with arsenic trioxide (As2O3) alone or in combination with arotinoid trometamol (retinoic acid receptor agonist). Flow cytometric analysis, PCR array and Western blot were used to determine the potential mechanism and signaling pathways associated with arsenic carcinogenesis. The results showed that low concentration As2O3 could stimulate keratinocyte proliferation, and arotinoid trometamol inhibited the process via regulating the expression of about 20 genes. These genes included components of Wnt signaling (CSNK1A1L, CTNNB1, SFRP1, Wnt10B, Wnt11, Wnt16, Wnt5A, Wnt8A), Shh signaling (C6orf138, HHIP, PTCHD1) and BMP signaling pathway (BMP2, BMP7). The changes of some differentially expressed genes of these signaling pathways in As2O3 treatment group were counteracted by the subsequent arotinoid trometamol treatment. Our data suggest that dysregulation and cross-talk of Wnt, Shh and BMP signals play great roles in the process of arsenic-induced carcinogenesis, which could be antagonized by arotinoid trometamol.

OBJECTIVE: To evaluate the association of single nucleotide polymorphisms (SNPs) in genes/loci consistently altered in nonsyndromic oral clefts in patients with oral and breast cancer in a Brazilian population.
DESIGN: This case-control study evaluated the association of SNPs in IRF6 (rs642961), WNT3A (rs708111), GSK3β (rs9879992), 8q24 (rs987525) and WNT11 (rs1533767), representing regions consistently identified as of susceptibility for oral clefts, with oral cancer (oral squamous cell carcinoma) and breast cancer. Logistic regression analyses were used for confounding adjustments, and p values ≤0.01 were considered statistically significant (Bonferroni correction = 0.05/5 polymorphic markers).
RESULTS: The minor G allele of rs9879992 in GSK3β was associated with oral cancer risk (p = 0.02), whereas rs1533767 in WNT11 showed a protective effect against it (p = 0.04). Several SNP-SNP interactions containing GSK3β rs9879992 were significantly associated with oral cancer after 1000 permutation test. To breast cancer, the A allele of rs987525 was associated with increase risk in early stage (p = 0.02) and SNP-SNP interactions involving the 5 SNPs were significantly observed, with the most significant interaction among rs708111, rs1533767, rs9879992 and rs642961 (p
CONCLUSION: Our results reveal associations of SNPs consistently altered in oral cleft with oral and breast cancer risk, raising interesting possibilities to identify risk markers for those tumors.

MicroRNAs (miRNAs) are solid factors involved in the initiation and progression of hepatocellular carcinoma (HCC). Recently, miR-298 is recognized as a cancer-associated miRNA in breast, gastric and ovarian cancer. However, the functional role of miR-298 and its underlying mechanism are rarely reported in HCC. Herein, we found that the expression of miR-298 was down-regulated in HCC tissues and cell lines. The in vitro experiments showed that miR-298 overexpression inhibited cell proliferation, migration and invasion, and induced G1 arrest and apoptosis of HCC cells. miR-298 knockdown exerted an opposite effect on these cellular behaviors of HCC cells. Moreover, miR-298 restoration suppressed HCC tumor growth and metastasis in vivo. Additionally, catenin delta 1 (CTNND1) was demonstrated to be a direct target of miR-298 in HCC cells. CTNND1 knockdown led to similar effects with miR-298 overexpression on HCC cell proliferation, cell cycle progression, apoptosis and mobility. CTNND1 restoration reversed miR-298-induced inhibitory effects on HCC cells. Mechanistically, both miR-298 overexpression and CTNND1 knockdown repressed Wnt/β-catenin signaling and resulted in reduced expression of β-catenin, WNT11, Cyclin D1 and MMP7 in HCCLM3 cells. While, CTNND1 restoration abolished miR-298-induced inactivation of Wnt/β-catenin signaling. In conclusion, our findings provide the first evidence that miR-298 suppresses HCC progression at least partially by targeting CTNND1-mediated Wnt/β-catenin signaling. MiR-298 may be a target for new therapies in HCC patients.

The present study investigated the role of Wnt11 in multicellular tumor spheroid-like structures (MCTS) ovarian cancer cell proliferation, migration and invasion in vitro and in vivo tumorigenesis and metastasis in xenograft nude mice model. Moreover, samples from human serous ovarian cancer (SOC) were used to assess the association of Wnt11 with integrins and cadherins. The data showed that Wnt11 overexpressing SKOV-3 cells became more compact accompanied by increased expression of E-and N-cadherin and lower expression of EpCAM and CD44. The α5, β2, β3 and β6 integrin subunits expression levels were significantly reduced in Wnt11 overexpressing cells accompanied with significantly reduced disaggregation of Wnt11 overexpressing SKOV-3 MCTS on ECM components. Moreover, Wnt11 overexpressing SKOV-3 MCTS showed decreased migration, invasion as well as no tumor growth and metastasis in vivo. We found that Wnt11 significantly and negatively correlated with ITGB2, ITGB6, and EpCAM and positively with CDH-1 in high-grade SOC specimens. Our results suggest that Wnt11 impedes MCTS attachment to ECM components and therefore can affect ovarian cancer progression.

Wnt-11 promotes cancer cell migration and invasion independently of β-catenin but the receptors involved remain unknown. Here, we provide evidence that FZD

Wnt-11 is a positive regulator of the Wnt signaling pathway, which plays a crucial role in carcinogenesis. However, Wnt-11 expression in cervical cancer has not been well investigated. The aim of this study was to investigate the role of Wnt-11 in cervical tumor proliferation and invasion. This study examined 24 normal cervical squamous epithelia, 29 cervical intraepithelial neoplasia (CIN), and 78 cervical cancer samples. The expression of Wnt-11 was investigated by immunohistochemistry and quantitative reverse transcription-polymerase chain reaction analysis. The expression of the high-risk human papilloma virus (HR-HPV) E6 oncoprotein was also investigated by immunohistochemistry. In addition, the expression of Wnt-11, HR-HPV E6, JNK-1, phosphorylated JNK-1(P-JNK1), and β-catenin was examined by western blot analysis following Wnt-11 knockdown or overexpression in HeLa or SiHa cells, respectively. The promotion of cervical cancer cell proliferation and invasion was investigated using the cell counting kit-8 and Matrigel invasion assay, respectively. Wnt-11 and HR-HPV E6 expression increased in a manner that corresponded with the progression of cervical cancer and was significantly correlated with the International Federation of Gynecology and Obstetrics cancer stage, lymph node metastasis, tumor size, and HPV infection. Wnt-11 protein expression was positively associated with HR-HPV E6 protein expression in all 78 cervical cancer samples (P < 0.001). Furthermore, Wnt-11 was positively associated with P-JNK1 expression and promoted cervical cancer cell proliferation and invasion. These observations suggest that the increased Wnt-11 expression observed in cervical cancer cells may lead to the phosphorylation and activation of JNK-1 and significantly promote tumor cell proliferation and cell migration/invasion through activation of the Wnt/JNK pathway. Consequently, Wnt-11 may serve as a novel target for cervical cancer therapy.

Microarray analysis revealed genes of the posterior HOXD locus normally involved in bone formation to be over-expressed in primary Ewing sarcoma (ES). The expression of posterior HOXD genes was not influenced via ES pathognomonic EWS/ETS translocations. However, knock down of the dickkopf WNT signaling pathway inhibitor 2 (DKK2) resulted in a significant suppression of HOXD10, HOXD11 and HOXD13 while over-expression of DKK2 and stimulation with factors of the WNT signaling pathway such as WNT3a, WNT5a or WNT11 increased their expression. RNA interference demonstrated that individual HOXD genes promoted chondrogenic differentiation potential, and enhanced expression of the bone-associated gene RUNX2. Furthermore, HOXD genes increased the level of the osteoblast- and osteoclast-specific genes, osteocalcin (BGLAP) and platelet-derived growth factor beta polypeptide (PDGFB), and may further regulate endochondral bone development via induction of parathyroid hormone-like hormone (PTHLH). Additionally, HOXD11 and HOXD13 promoted contact independent growth of ES, while in vitro invasiveness of ES lines was enhanced by all 3 HOXD genes investigated and seemed mediated via matrix metallopeptidase 1 (MMP1). Consequently, knock down of HOXD11 or HOXD13 significantly suppressed lung metastasis in a xeno-transplant model in immune deficient mice, providing overall evidence that posterior HOXD genes promote clonogenicity and metastatic potential of ES.

Androgen deprivation therapy (ADT) is the standard of care for metastatic prostate cancer and initially induces tumor regression, but invariably results in castration-resistant prostate cancer through various mechanisms, incompletely discovered. Our aim was to analyze the dynamic modulation, determined by ADT, of the expression of selected genes involved in the pathogenesis and progression of prostate cancer (TMPRSS2:ERG, WNT11, SPINK1, CHGA, AR, and SPDEF) using real-time polymerase chain reaction in a series of 59 surgical samples of prostate carcinomas, including 37 cases preoperatively treated with ADT and 22 untreated cases, and in 43 corresponding biopsies. The same genes were analyzed in androgen-deprived and control LNCaP cells. Three genes were significantly up-modulated (WNT11 and AR) or down-modulated (SPDEF) in patients treated with ADT versus untreated cases, as well as in androgen-deprived LNCaP cells. The effect of ADT on CHGA gene up-modulation was almost exclusively detected in cases positive for the TMPRSS2:ERG fusion. The correlation between biopsy and surgical samples was poor for most of the tested genes. Gene expression analysis of separate tumor areas from the same patient showed an extremely heterogeneous profile in the 6 tested cases (all untreated). In conclusion, our results strengthened the implication of ADT in promoting a prostate cancer aggressive phenotype and identified potential biomarkers, with special reference to the TMPRSS2:ERG fusion, which might favor the development of neuroendocrine differentiation in hormone-treated patients. However, intratumoral heterogeneity limits the use of gene expression analysis as a potential prognostic or predictive biomarker in patients treated with ADT.

BACKGROUND: Increasing evidence supports the association of CTNND1 with tumor development and progression. However, the mechanism and clinical significance of CTNND1 deregulation in hepatocellular carcinoma (HCC) remains unknown. In this study, we aim to investigate the role of CTNND1 in HCC.
METHODS: qRT-PCR and immunohistochemical analyses were used to measure the levels of CTNND1 in HCC specimens and HCC cell lines. CTNND1 and shCTNND1 were transfected into HCC cell lines to investigate its role in HCC. Cell migration and invasion were measured by Transwell and Matrigel analyses in vitro. In vivo metastasis assays were performed in SCID mice.
RESULTS: In clinical HCC samples, we found that CTNND1 expression was significantly up-regulated in cancer lesions compared with paired normal liver tissues. By silencing or overexpressing CTNND1 in HCC cells, we found that CTNND1 could promote cell proliferation, migration, and invasion in vitro. An in-vivo assay showed that CTNND1 dramatically promoted HCC cell tumor formation and metastasis. Moreover, CTNND1 promoted HCC metastasis, at least in part, by indirectly enhancing Wnt/β-catenin signaling. Consistent with these results, the expression of CTNND1 was positively correlated with β-catenin, WNT11, Cyclin D1, and BMP7 expression in human HCC specimens.
CONCLUSIONS: Our study provides evidence that CTNND1 functions as a novel tumor oncogene in HCC, and may be a potential therapeutic target for HCC management.

Changes in cellular oxygen tension play important roles in physiological processes including development and pathological processes such as tumor promotion. The cellular adaptations to sustained hypoxia are mediated by hypoxia-inducible factors (HIFs) to regulate downstream target gene expression. With hypoxia, the stabilized HIF-α and aryl hydrocarbon receptor nuclear translocator (ARNT, also known as HIF-β) heterodimer bind to hypoxia response elements (HREs) and regulate expression of target genes. Here, we report that WNT11 is induced by hypoxia in many cell types, and that transcription of WNT11 is regulated primarily by HIF-1α. We observed induced WNT11 expression in the hypoxic area of allograft tumors. In addition, in mice bearing orthotopic malignant gliomas, inhibition with bevacizumab of vascular endothelial growth factor, which is an important stimulus for angiogenesis, increased nuclear HIF-1α and HIF-2α, and expression of WNT11. Gain- and loss-of-function approaches revealed that WNT11 stimulates proliferation, migration and invasion of cancer-derived cells, and increases activity of matrix metalloproteinase (MMP)-2 and 9. Since tumor hypoxia has been proposed to increase tumor aggressiveness, these data suggest WNT11 as a possible target for cancer therapies, especially for tumors treated with antiangiogenic therapy.

The ESRRA gene encodes a transcription factor and regulates several genes, such as WNT11 and OPN, involved in tumorigenesis. It is upregulated in several cancers, including OSCC. We have previously shown that the tumor suppressor miR-125a targets ESRRA, and its downregulation causes upregulation of ESRRA in OSCC. Upregulation of ESRRA in the absence of downregulation of miR-125a in a subset of OSCC samples suggests the involvement of an alternative mechanism. Using TaqMan(®) copy number assay, here we report for the first time that the genomic amplification of ESRRA causes its upregulation in a subset of OSCC samples. Ectopic overexpression of ESRRA led to accelerated cell proliferation, anchorage-independent cell growth and invasion, and inhibited apoptosis. Whereas, knockdown of ESRRA expression by siRNA led to reduced cell proliferation, anchorage-independent cell growth and invasion, and accelerated apoptosis. Furthermore, the delivery of a synthetic biostable ESRRA siRNA to OSCC cells resulted in regression of xenografts in nude mice. Thus, the genomic amplification of ESRRA is another novel mechanism for its upregulation in OSCC. Based on our in vitro and in vivo experiments, we suggest that targeting ESRRA by siRNA could be a novel therapeutic strategy for OSCC and other cancers.

E-cadherin inactivation underpins the progression of invasive lobular breast carcinoma (ILC). In ILC, p120-catenin (p120) translocates to the cytosol where it controls anchorage independence through the Rho-Rock signaling pathway, a key mechanism driving tumor growth and metastasis. We now demonstrate that anchorage-independent ILC cells show an increase in nuclear p120, which results in relief of transcriptional repression by Kaiso. To identify the Kaiso target genes that control anchorage independence we performed genome-wide mRNA profiling on anoikis-resistant mouse ILC cells, and identified 29 candidate target genes, including the established Kaiso target Wnt11. Our data indicate that anchorage-independent upregulation of Wnt11 in ILC cells is controlled by nuclear p120 through inhibition of Kaiso-mediated transcriptional repression. Finally, we show that Wnt11 promotes activation of RhoA, which causes ILC anoikis resistance. Our findings thereby establish a mechanistic link between E-cadherin loss and subsequent control of Rho-driven anoikis resistance through p120- and Kaiso-dependent expression of Wnt11.

BACKGROUND: Over 70% of cancer metastasis from prostate cancer develops bone metastases that are not sensitive to hormonal therapy, radiation therapy, or chemotherapy. The epithelial-to-mesenchymal transition (EMT) genetic program is implicated as a significant contributor to prostate cancer progression. As such, targeting the EMT represents an important therapeutic strategy for preventing or treating prostate cancer metastasis. Berberine is a natural alkaloid with significant antitumor activities against many types of cancer cells. In this study, we investigated the molecular mechanism by which berberine represses the metastatic potential of prostate cancer.
METHODS: The effects of berberine on cell migration and invasion were determined by transwell migration assay and Matrigel invasion assay. Expressions of EMT-related genes were determined by an EMT PCR Array and a quantitative RT-PCR. The prognostic relevance of berberine's modulation of EMT-related genes in prostate cancer was evaluated using Kaplan-Meier survival analysis.
RESULTS: Berberine exerted inhibitory effects on the migratory and invasive abilities of highly metastatic prostate cancer cells. These inhibitory effects of berberine resulted in significant repression of a panel of mesenchymal genes that regulate the developmental EMT. Among EMT-related genes downregulated by berberine, high BMP7, NODAL and Snail gene expressions of metastatic prostate cancer tissues were associated with shorter survival of prostate cancer patients and provide potential therapeutic interventions.
CONCLUSIONS: We concluded that berberine should be developed as a pharmacological agent for use in combination with other anticancer drug for treating metastatic prostate cancer.

BACKGROUND: One of the most insidious characteristics of cancer is its spread to and ability to compromise distant organs via the complex process of metastasis. Communication between cancer cells and organ-resident cells via cytokines/chemokines and direct cell-cell contacts are key steps for survival, proliferation and invasion of metastasized cancer cells in organs. Precision-cut liver slices (PCLS) are considered to closely reflect the in vivo situation and are potentially useful for studying the interaction of cancer cells with liver-resident cells as well as being a potentially useful tool for screening anti-cancer reagents. Application of the PCLS technique in the field of cancer research however, has not yet been well developed.
RESULTS: We established the mouse PCLS system using perfluorodecalin (PFD) as an artificial oxygen carrier. Using this system we show that the adherence of green fluorescent protein (GFP) labeled MDA-MB-231 (highly invasive) cells to liver tissue in the PCLS was 5-fold greater than that of SK-BR-3 (less invasive) cells. In addition, we generated PCLS from THOC5, a member of transcription/export complex (TREX), knockout (KO) mice. The PCLS still expressed Gapdh or Albumin mRNAs at normal levels, while several chemokine/growth factor or metalloprotease genes, such as Cxcl12, Pdgfa, Tgfb, Wnt11, and Mmp1a genes were downregulated more than 2-fold. Interestingly, adhesion of cancer cells to THOC5 KO liver slices was far less (greater than 80% reduction) than to wild-type liver slices.
CONCLUSION: Mouse PCLS cultures in the presence of PFD may serve as a useful tool for screening local adherence and invasiveness of individual cancer cells, since single cells can be observed. This method may also prove useful for identification of genes in liver-resident cells that support cancer invasion by using PCLS from transgenic liver.

The Notch signaling pathway plays versatile roles during heart development. However, there is contradictory evidence that Notch pathway either facilitates or impairs cardiomyogenesis in vitro. In this study, we developed iPSCs by reprogramming of murine fibroblasts with GFP expression governed by Oct4 promoter, and identified an effective strategy to enhance cardiac differentiation through timely modulation of Notch signaling. The Notch inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester) alone drove the iPSCs to a neuronal fate. After mesoderm induction of embryoid bodies initiated by ascorbic acid (AA), the subsequent treatment of DAPT accelerated the generation of spontaneously beating cardiomyocytes. The timed synergy of AA and DAPT yielded an optimal efficiency of cardiac differentiation. Mechanistic studies showed that Notch pathway plays a biphasic role in cardiomyogenesis. It favors the early-stage cardiac differentiation, but exerts negative effects on the late-stage differentiation. Therefore, DAPT administration at the late stage enforced the inhibition of endogenous Notch activity, thereby enhancing cardiomyogenesis. In parallel, DAPT dramatically augmented the expression of Wnt3a, Wnt11, BMP2, and BMP4. In conclusion, our results highlight a practicable approach to generate cardiomyocytes from iPSCs based on the stage-specific biphasic roles of Notch signaling in cardiomyogenesis.

Estrogen-related receptor α (ESRRA) functions as a transcription factor and regulates the expression of several genes, such as WNT11 and OPN. Up-regulation of ESRRA has been reported in several cancers. However, the mechanism underlying its up-regulation is unclear. Furthermore, the reports regarding the role and regulation of ESRRA in oral squamous cell carcinoma (OSCC) are completely lacking. Here, we show that tumor suppressor miR-125a directly binds to the 3'UTR of ESRRA and represses its expression. Overexpression of miR-125a in OSCC cells drastically reduced the level of ESRRA, decreased cell proliferation, and increased apoptosis. Conversely, the delivery of an miR-125a inhibitor to these cells drastically increased the level of ESRRA, increased cell proliferation, and decreased apoptosis. miR-125a-mediated down-regulation of ESRRA impaired anchorage-independent colony formation and invasion of OSCC cells. Reduced cell proliferation and increased apoptosis of OSCC cells were dependent on the presence of the 3'UTR in ESRRA. The delivery of an miR-125a mimic to OSCC cells resulted in marked regression of xenografts in nude mice, whereas the delivery of an miR-125a inhibitor to OSCC cells resulted in a significant increase of xenografts and abrogated the tumor suppressor function of miR-125a. We observed an inverse correlation between the expression levels of miR-125a and ESRRA in OSCC samples. In summary, up-regulation of ESRRA due to down-regulation of miR-125a is not only a novel mechanism for its up-regulation in OSCC, but decreasing the level of ESRRA by using a synthetic miR-125a mimic may have an important role in therapeutic intervention of OSCC and other cancers.

Here we intend to identify key genes and pathways in the pathogenesis of colorectal cancer (CRC) through analyzing microarray data with bioinformatic tools. The gene expression profile dataset GSE23878 was downloaded from Gene Expression Omnibus and differentially expressed genes (DEGs) were screened out using Student's t-test. GO function and KEGG pathway enrichment analyses were performed for these DEGs with the DAVID online tool. Interaction network was constructed among the over-represented pathways based on the protein-protein interactions within the pathways. Besides, the protein interaction information obtained from HPRD database were applied to constructed protein-protein interaction networks among the DEGs and hub genes and function module were screened out. A total of 2,296 DEGs were obtained and they were enriched in 34 pathways. An interaction network was constructed among 32 pathways, in which p53 signaling pathway acted as the hub pathway as it showed the highest node degree. The protein-protein interaction network comprised 1,481 interaction relationships among 332 genes which included 40 DEGs. Further analysis revealed that theses DEGs formed 7 function modules and many genes, such as PDGFRB, MET, FZD2, CCND1, PRKCB, ARHGEF6, JUP, WNT2, WNT5A and WNT11 were key genes in the networks. The DEGs and disturbed biological functions uncovered in present study may play important roles in the development of CRC and can contribute to the understanding on molecular mechanisms of CRC. Further these DEGs we obtained can be acted as potential biomarkers for diagnosis and therapy of CRC.

Uterine leiomyomas are extremely common estrogen and progesterone-dependent tumors of the myometrium and cause irregular uterine bleeding, severe anemia, and recurrent pregnancy loss in 15-30% of reproductive-age women. Each leiomyoma is thought to arise from a single mutated myometrial smooth muscle stem cell. Leiomyoma side-population (LMSP) cells comprising 1% of all tumor cells and displaying tumor-initiating stem cell characteristics are essential for estrogen- and progesterone-dependent in vivo growth of tumors, although they have remarkably lower estrogen/progesterone receptor levels than mature myometrial or leiomyoma cells. However, how estrogen/progesterone regulates the growth of LMSP cells via mature neighboring cells is unknown. Here, we demonstrate a critical paracrine role of the wingless-type (WNT)/β-catenin pathway in estrogen/progesterone-dependent tumorigenesis, involving LMSP and differentiated myometrial or leiomyoma cells. Estrogen/progesterone treatment of mature myometrial cells induced expression of WNT11 and WNT16, which remained constitutively elevated in leiomyoma tissues. In LMSP cells cocultured with mature myometrial cells, estrogen-progesterone selectively induced nuclear translocation of β-catenin and induced transcriptional activity of its heterodimeric partner T-cell factor and their target gene AXIN2, leading to the proliferation of LMSP cells. This effect could be blocked by a WNT antagonist. Ectopic expression of inhibitor of β-catenin and T-cell factor 4 in LMSP cells, but not in mature leiomyoma cells, blocked the estrogen/progesterone-dependent growth of human tumors in vivo. We uncovered a paracrine role of the WNT/β-catenin pathway that enables mature myometrial or leiomyoma cells to send mitogenic signals to neighboring tissue stem cells in response to estrogen and progesterone, leading to the growth of uterine leiomyomas.

INTRODUCTION: Canonical and non-canonical Wnt pathways are involved in the genesis of multiple tumors; however, their role in pituitary tumorigenesis is mostly unknown.
OBJECTIVE: This study evaluated gene and protein expression of Wnt pathways in pituitary tumors and whether these expression correlate to clinical outcome.
MATERIALS AND METHODS: Genes of the WNT canonical pathway: activating ligands (WNT11, WNT4, WNT5A), binding inhibitors (DKK3, sFRP1), β-catenin (CTNNB1), β-catenin degradation complex (APC, AXIN1, GSK3β), inhibitor of β-catenin degradation complex (AKT1), sequester of β-catenin (CDH1), pathway effectors (TCF7, MAPK8, NFAT5), pathway mediators (DVL-1, DVL-2, DVL-3, PRICKLE, VANGL1), target genes (MYB, MYC, WISP2, SPRY1, TP53, CCND1); calcium dependent pathway (PLCB1, CAMK2A, PRKCA, CHP); and planar cell polarity pathway (PTK7, DAAM1, RHOA) were evaluated by QPCR, in 19 GH-, 18 ACTH-secreting, 21 non-secreting (NS) pituitary tumors, and 5 normal pituitaries. Also, the main effectors of canonical (β-catenin), planar cell polarity (JNK), and calcium dependent (NFAT5) Wnt pathways were evaluated by immunohistochemistry.
RESULTS: There are no differences in gene expression of canonical and non-canonical Wnt pathways between all studied subtypes of pituitary tumors and normal pituitaries, except for WISP2, which was over-expressed in ACTH-secreting tumors compared to normal pituitaries (4.8x; p = 0.02), NS pituitary tumors (7.7x; p = 0.004) and GH-secreting tumors (5.0x; p = 0.05). β-catenin, NFAT5 and JNK proteins showed no expression in normal pituitaries and in any of the pituitary tumor subtypes. Furthermore, no association of the studied gene or protein expression was observed with tumor size, recurrence, and progressive disease. The hierarchical clustering showed a regular pattern of genes of the canonical and non-canonical Wnt pathways randomly distributed throughout the dendrogram.
CONCLUSIONS: Our data reinforce previous reports suggesting no activation of canonical Wnt pathway in pituitary tumorigenesis. Moreover, we describe, for the first time, evidence that non-canonical Wnt pathways are also not mis-expressed in the pituitary tumors.

PURPOSE: Patients with triple-negative breast cancer (TNBC) and residual disease after neoadjuvant chemotherapy generally have worse outcome; however, some patients with residual tumor after neoadjuvant chemotherapy do not relapse. We hypothesize that there are subgroups of patients with chemoresistant TNBC with different prognosis.
EXPERIMENTAL DESIGN: Forty-nine chemoresistant cases from 111 patients with TNBC treated with neoadjuvant chemotherapy (M.D. Anderson Cancer Center, Houston, TX) constituted the discovery cohort, and 25 chemoresistant samples from 47 neoadjuvant chemotherapy-treated TNBC (The Methodist Hospital, Houston, TX) were chosen for validation. Extended validation was carried out in 269 operable TNBC predicted to be chemoresistant by expression pattern from published datasets.
RESULTS: We established a seven-gene prognostic signature using dChip and gene set enrichment analyses. In the independent validation cohort, the classifier predicted correctly with positive predictive value of 75.0% and negative predictive value (i.e., relapse-free survival; RFS) of 76.9% at 3 years. Those predicted to relapse had a HR of 4.67 [95% confidence interval (CI): 1.27-17.15] for relapse in 3 years. In extended validation, patients predicted not to relapse exhibited 3-year RFS of 78.9%, whereas the 3-year RFS was 48.5% for patients predicted to relapse, with HR of 2.61 (95% CI: 1.52-4.49). The TNBC subgroup that predicted to have relatively favorable prognosis was characterized by high expression of "luminal-like" genes [androgen-receptor (AR) and GATA3], whereas the subgroup with worse prognosis was characterized by expression of cancer stem-cell markers.
CONCLUSION: We developed a clinically relevant signature for patients with chemoresistant TNBC. For these women, new therapeutic strategies like targeting AR activation or cancer stem cells may need to be developed.

The majority of lung cancers (LC) belong to the non-small cell lung carcinoma (NSCLC) type. The two main NSCLC sub-types, namely adenocarcinoma (AC) and squamous cell carcinoma (SCC), respond differently to therapy. Whereas the link between cigarette smoke and lung cancer risk is well established, the relevance of non-canonical Wnt pathway up-regulation detected in SCC remains poorly understood. The present study was undertaken to investigate further the molecular events in canonical and non-canonical Wnt signalling during SCC development. A total of 20 SCC and AC samples with matched non-cancerous controls were obtained after surgery. TaqMan array analysis confirmed up-regulation of non-canonical Wnt5a and Wnt11 and identified down-regulation of canonical Wnt signalling in SCC samples. The molecular changes were tested in primary small airway epithelial cells (SAEC) and various lung cancer cell lines (e.g. A549, H157, etc). Our studies identified Wnt11 and Wnt5a as regulators of cadherin expression and potentiated relocation of β-catenin to the nucleus as an important step in decreased cellular adhesion. The presented data identifies additional details in the regulation of SCC that can aid identification of therapeutic drug targets in the future.

Endometrial cancer is the most common gynecological malignancy, with more than 280,000 cases occurring annually worldwide. Although previous studies have identified important common somatic mutations in endometrial cancer, they have primarily focused on a small set of known cancer genes and have thus provided a limited view of the molecular basis underlying this disease. Here we have developed an integrated systems-biology approach to identifying novel cancer genes contributing to endometrial tumorigenesis. We first performed whole-exome sequencing on 13 endometrial cancers and matched normal samples, systematically identifying somatic alterations with high precision and sensitivity. We then combined bioinformatics prioritization with high-throughput screening (including both shRNA-mediated knockdown and expression of wild-type and mutant constructs) in a highly sensitive cell viability assay. Our results revealed 12 potential driver cancer genes including 10 tumor-suppressor candidates (ARID1A, INHBA, KMO, TTLL5, GRM8, IGFBP3, AKTIP, PHKA2, TRPS1, and WNT11) and two oncogene candidates (ERBB3 and RPS6KC1). The results in the "sensor" cell line were recapitulated by siRNA-mediated knockdown in endometrial cancer cell lines. Focusing on ARID1A, we integrated mutation profiles with functional proteomics in 222 endometrial cancer samples, demonstrating that ARID1A mutations frequently co-occur with mutations in the phosphatidylinositol 3-kinase (PI3K) pathway and are associated with PI3K pathway activation. siRNA knockdown in endometrial cancer cell lines increased AKT phosphorylation supporting ARID1A as a novel regulator of PI3K pathway activity. Our study presents the first unbiased view of somatic coding mutations in endometrial cancer and provides functional evidence for diverse driver genes and mutations in this disease.

ERRα is an orphan nuclear receptor emerging as a novel biomarker of breast cancer. Over-expression of ERRα in breast tumor is considered as a prognostic factor of poor clinical outcome. The mechanisms underlying the dysexpression of this nuclear receptor, however, are poorly understood. MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level and play important roles in tumor initiation and progression. In the present study, we have identified that the expression of ERRα is regulated by miR-137, a potential tumor suppressor microRNA. The bioinformatics search revealed two putative and highly conserved target-sites for miR-137 located within the ERRα 3'UTR at nt 480-486 and nt 596-602 respectively. Luciferase-reporter assay demonstrated that the two predicted target sites were authentically functional. They mediated the repression of reporter gene expression induced by miR-137 in an additive manner. Moreover, ectopic expression of miR-137 down-regulated ERRα expression at both protein level and mRNA level, and the miR-137 induced ERRα-knockdown contributed to the impaired proliferative and migratory capacity of breast cancer cells. Furthermore, transfection with miR-137 mimics suppressed at least two downstream target genes of ERRα-CCNE1 and WNT11, which are important effectors of ERRα implicated in tumor proliferation and migration. Taken together, our results establish a role of miR-137 in negatively regulating ERRα expression and breast cancer cell proliferation and migration. They suggest that manipulating the expression level of ERRα by microRNAs has the potential to influence breast cancer progression.

BACKGROUND: Multistep pathways and mechanisms are involved in the development of oral cancer. Chromosomal alterations are one of such key mechanisms implicated oral carcinogenesis. Therefore, this study aims to determine the genomic copy number alterations (CNAs) in oral squamous cell carcinoma (OSCC) using array comparative genomic hybridization (aCGH) and in addition attempt to correlate CNAs with modified gene expression.
MATERIALS AND METHODS: Genome-wide screening was performed on 15 OSCCs using high-density aCGH. On the basis of pathway analysis, three genes (ISG15, Nestin and WNT11) which mapped to CNA regions were selected for further evaluation of their mRNA expression using quantitative reverse transcriptase PCR (qRT-PCR).
RESULTS: Copy number alterations were observed on multiple genomic regions, including amplifications on 1p, 3q, 5p, 6p, 7p, 8q, 9q, 11q, 12q, 16p, 18p and deletions on 3p, 7q, 8p, 11q, 19q and 20q. Among the three selected genes, ISG15 had the highest mRNA expression level with a 22.5-fold increase, followed by Nestin with a 4.5-fold increase and WNT11 with a 2.5-fold increase.
CONCLUSIONS: This study has identified several major CNAs in oral cancer genomes and indicated that this correlates with over expression of the ISG15, WNT11, and Nestin genes.

Our previous report revealed that the expression of Frizzled-7 (FZD7) in colorectal cancer (CRC) and its possible role in CRC progression. In this study we measured the expression levels of candidate FZD7 ligands, Wnt3 and Wnt11 in colon cancer cell lines (n = 7) and primary CRC tissues (n = 133) by quantitative RT-PCR. We also examined the functional effects of Wnt11 with the use of Wnt11 transfectants of colon cancer HCT-116 cells. Wnt11 transfectants showed the increased proliferation and migration/invasion activities compared to mock cells. Western blot analysis of transfectants revealed that phosphorylation of JNK and c-jun was increased after Wnt11 transfection. Wnt11 mRNA expression was significantly higher in the stage I, II, III, or IV tumor tissues than in non-tumor tissues (overall P < 0.003), while there was no significant difference in Wnt3 mRNA expression between tumor and non-tumor tissues. In addition, Wnt11 mRNA expression was significantly higher in patients with recurrence or death after surgery than in those with no recurrence (disease free) after surgery (P = 0.018). We also compared the expression levels of Wnt11 mRNA with those of FZD7 mRNA in the same CRC samples. Wnt11 mRNA expression was significantly higher in patients with higher FZD7 mRNA levels than in those with lower FZD7 mRNA levels (P = 0.0005). The expression levels of Wnt11 mRNA were correlated with those of FZD7 mRNA (P < 0.0001). These data suggest that Wnt11 may play an important role in CRC progression.

BACKGROUND: Canonical and non-canonical Wnt signaling pathways modulate diverse cellular processes during embryogenesis and post-natally. Their deregulations have been implicated in cancer development and progression. Wnt signaling is essential for odontogenesis. The ameloblastoma is an odontogenic epithelial neoplasm of enamel organ origin. Altered expressions of Wnts-1, -2, -5a, and -10a are detected in this tumor. The activity of other Wnt members remains unclarified.
MATERIALS AND METHODS: Canonical (Wnts-1, -2, -3, -8a, -8b, -10a, and -10b), non-canonical (Wnts-4, -5a, -5b, -6, 7a, -7b, and -11), and indeterminate groups (Wnts-2b and -9b) were examined immunohistochemically in 72 cases of ameloblastoma (19 unicystic [UA], 35 solid/multicystic [SMA], eight desmoplastic [DA], and 10 recurrent [RA]).
RESULTS: Canonical Wnt proteins (except Wnt-10b) were heterogeneously expressed in ameloblastoma. Their distribution patterns were distinctive with some overlap. Protein localization was mainly membranous and/or cytoplasmic. Overexpression of Wnt-1 in most subsets (UA = 19/19; SMA = 35/35; DA = 5/8; RA = 7/10) (P < 0.05), Wnt-3 in granular cell variant (n = 3/3), and Wnt-8b in DA (n = 8/8) was key observations. Wnts-8a and -10a demonstrated enhanced expression in tumoral buddings and acanthomatous areas. Non-canonical and indeterminate Wnts were absent except for limited Wnt-7b immunoreactivity in UA (n = 1/19) and SMA (n = 1/35). Stromal components expressed variable Wnt positivity.
CONCLUSION: Differential expression of Wnt ligands in different ameloblastoma subtypes suggests that the canonical and non-canonical Wnt pathways are selectively activated or repressed depending on the tumor cell differentiation status. Canonical Wnt pathway is most likely the main transduction pathway while Wnt-1 might be the key signaling molecule involved in ameloblastoma tumorigenesis.

Genetic studies of Wnt11 have revealed many insights into the roles and regulation of Wnt11, particularly during development. New tools to study Wnt11 have recently become available, making it timely to review the literature regarding this unique Wnt family member. In this study, we focus on mammalian Wnt11, describing its main sites of expression during development, and how the Wnt11 gene is regulated. We highlight an emerging theme in which canonical Wnt signals regulate Wnt11 expression through transcription factors in addition to, or other than, Tcf/LEF family members. We also discuss the frizzled family and other receptors that bind to Wnt11, the intracellular kinases and small GTPases that act downstream of Wnt11, and the effects of Wnt11 on Wnt/β-catenin signalling. Finally, we elaborate on the relevance of Wnt11 to human cancer, where it appears to be important both for proliferation and/or survival during normal differentiation and for migration/invasion.

Oral squamous cell carcinoma (OSCC) accounts for more than 90% of the malignant neoplasms that arise in the mucosa of the upper aerodigestive tract. Recent studies of cleft lip/palate have shown the association of genes involved in cancer. WNT pathway genes have been associated with several types of cancer and recently with cleft lip/palate. To investigate if genes associated with cleft lip/palate were also associated with oral cancer, we genotyped 188 individuals with OSCC and 225 control individuals for markers in AXIN2, AXIN1, GSK3β, WNT3A, WNT5A, WNT8A, WNT11, WNT3, and WNT9B. Statistical analysis was performed with PLINK 1.06 software to test for differences in allele frequencies of each polymorphism between cases and controls. We found association of SNPs in GSK3B (p = 0.0008) and WNT11 (p = 0.03) with OSCC. We also found overtransmission of GSK3B haplotypes in OSCC cases. Expression analyses showed up-regulation of WNT3A, GSK3B, and AXIN1 and down-regulation of WNT11 in OSCC in comparison with control tissues (P < 0.001). Additional studies should focus on the identification of potentially functional variants in these genes as contributors to human clefting and oral cancer.

Peutz-Jeghers syndrome (PJS) is a rare, inherited disease caused by germline mutation of the LKB1 gene. Patients with PJS develop characteristic polyps in the digestive tract and carry an elevated risk of cancers in multiple organs, including the intestinal tract. While LKB1 is capable of phosphorylating AMPK and regulates the mTOR pathway, it is also known to be a multitasking protein that can influence other cellular processes, including cell polarity. We hypothesized that there may be other biological pathways directly or indirectly affected by the loss of LKB1 in PJS and aimed to investigate this possibility through transcriptional profiling of polyps harvested from an Lkb1(+/-) mouse model of PJS and from PJS patients. We identified alterations in the mRNA level of a wide range of genes, including some that are involved in Wnt signalling (Wnt5a, Wif1, Dixdc1, Wnt11, Ccnd1, and Ccnd2), although we did not observe nuclear localization of β-catenin in over 93 human PJS intestinal polyps or in 24 gastric polyps from Lkb1(+/-) mice. Among these genes, WNT5A, a non-canonical and non-transforming Wnt, is consistently up-regulated in both Lkb1(+/-) mice and human PJS polyps at a high level. We performed in situ hybridization to further define the spatial expression pattern of WNT5A and observed a strong signal in the stroma of mouse and human polyps compared to no or very low expression in the mucosa. Our findings indicate that WNT5A plays an important role in PJS polyposis.

E26 transforming sequence-related gene (ERG) is a transcription factor involved in normal hematopoiesis and is dysregulated in leukemia. ERG mRNA overexpression was associated with poor prognosis in a subset of patients with T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). Herein, a genome-wide screen of ERG target genes was conducted by chromatin immunoprecipitation-on-chip (ChIP-chip) in Jurkat cells. In this screen, 342 significant annotated genes were derived from this global approach. Notably, ERG-enriched targets included WNT signaling genes: WNT11, WNT2, WNT9A, CCND1 and FZD7. Furthermore, chromatin immunoprecipitation (ChIP) of normal and primary leukemia bone marrow material also confirmed WNT11 as a target of ERG in six of seven patient samples. A larger sampling of patient diagnostic material revealed that ERG and WNT11 mRNA were co-expressed in 80% of AML (n=30) and 40% in T-ALL (n=30) bone marrow samples. Small interfering RNA (siRNA)-mediated knockdown of ERG confirmed downregulation of WNT11 transcripts. Conversely, in a tet-on ERG-inducible assay, WNT11 transcripts were co-stimulated. A WNT pathway agonist, 6-bromoindirubin-3-oxime (BIO), was used to determine the effect of cell growth on the ERG-inducible cells. The addition of BIO resulted in an ERG-dependent proliferative growth advantage over ERG-uninduced cells. Finally, ERG induction prompted morphological transformation whereby round unpolarized K562 cells developed elongated protrusions and became polarized. This morphological transformation could effectively be inhibited with BIO and with siRNA knockdown of WNT11. In conclusion, ERG transcriptional networks in leukemia converge on WNT signaling targets. Specifically, WNT11 emerged as a direct target of ERG. Potent ERG induction promoted morphological transformation through WNT11 signals. The findings in this study unravel new ERG-directed molecular signals that may contribute to the resistance of current therapies in acute leukemia patients with poor prognosis characterized by high ERG mRNA expression.