Non-canonical Wnt Pathway Research Articles

Abstract 3306: Wnt pathway dynamics in breast cancer progression

Abstract Background: The canonical Wnt/β-catenin signaling pathway directs self-renewal cues for stem cells in multiple tissues. Through alternative receptors, noncanonical Wnt/β-catenin-independent pathways exist, mediating cellular processes that include planar cell polarity, convergent extension, actin/cytoskeletal rearrangements, and in some contexts, antagonism of canonical Wnt signaling. How different Wnt pathways cooperate within heterogeneous cellular landscapes in breast cancers remains an outstanding question. Within the current study, we investigated the role of the alternative Wnt receptor, Ror2, in mediating Wnt/β-catenin-independent functions during tumor progression. Experimental design and methods: A TP53 null transplantable GEM model was used in the current study, which exhibits subtype-specific molecular signatures characteristic of human breast cancers. A lentiviral-based shRNA strategy was developed for gene silencing of noncanonical Wnt pathway components in vivo, with emphasis on the alternative Wnt receptor, Ror2. This strategy was combined with pathway-specific Wnt reporters to survey the interplay of canonical and noncanonical Wnt pathways in vivo. Results: TP53 null mammary tumors differentially expressed the noncanonical Wnt receptor Ror2, with the highest levels of expression evident in basal-like tumors relative to luminal and claudin-low subtypes analyzed. Basal-like tumors also displayed Wnt/β-catenin active populations, which were inversely correlated with Ror2 expression. Lentiviral shRNA silencing of Ror2 within 3 independent basal-like models (T1, T2, 2225L) resulted in elevated Wnt/β-catenin activity along with various phenotypic outcomes, including squamous differentiation. Ror2, therefore, functions to restrain Wnt/β-catenin-dependent signaling in vivo. Despite the similarity in Wnt/β-catenin-dependent signaling outcome upon Ror2 depletion, gene expression profiling of Ror2-intact (shLUC) vs. Ror2-depleted (shRor2) tumors unveiled diverse gene expression changes within basal-like tumor models. Intriguingly, Ror2 silencing within 2225L basal-like tumors resulted in a change in the composition of tumor cell subpopulations. Additionally, gene expression analysis of shLUC vs. shRor2 tumors revealed that genes upregulated in shRor2 tumors were associated with claudin-low features, while genes downregulated were associated with basal-like features, reflecting changes in the cellular landscape upon Ror2 loss. Gene ontology analysis also revealed alterations in actin/cytoskeletal- and cell adhesion-associated genes upon Ror2 depletion. Conclusion: The current study highlights the differential effects of Ror2 loss within TP53 null basal-like models. In particular, Ror2 can regulate the plasticity between basal-like and claudin-low states, impacting cytoskeletal and cell adhesion dynamics. Current efforts are focused on how these changes impact tumor progression and metastasis. Citation Format: Kevin Roarty, Adam D. Pfefferle, Chad J. Creighton, Charles M. Perou, Jeffrey M. Rosen. Wnt pathway dynamics in breast cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3306.

Abstract 2515: Pharmacological WNT-inhibition acts synergistically with chemo- and radiotherapy by overcoming treatment-resistance in glioma stem cells

Abstract Objective: Glioma Stem Cells (GSCs) are considered to be responsible for Glioblastoma's (GBM’s) dismal prognosis. WNT signaling is one of the major players in GSCs that promotes their radio- and chemoresistance. In this study we tested whether LGK974, an inhibitor of both canonical and non-canonical WNT signaling, might sensitize GBM cells to chemotherapeutics and irradiation and determined possible mechanisms causing treatment-resistance in GBM. Methods: At first we analyzed MGMT promoter methylation status of several GBM cell lines in order to define cells resistant to the standard chemotherapeutic drug Temozolomide (TMZ). For further investigations we chose two cell lines resistant or sensitive to TMZ, respectively. IC50 concentrations of TMZ and LGK974 as well as IC50 dose of γ-irradiation were tested via Titer Blue cell viability assay. Effectiveness of different concentration of each single therapy and its combination with LGK974 was determined to create a drug-response-curve due to the computerized simulation of synergism and antagonism in drug combination studies (Chou, 2006). A reporter assay for canonical WNT/β-catenin signaling was used to quantify the effect of TMZ and irradiation alone and in combination with LGK974 on the canonical and non-canonical WNT pathway. Western Blot and qPCR were applied to analyze the effects of combinatory therapy on protein and gene expression concerning WNT pathway activation. Results: We observed a significant synergy of combined irradiation/TMZ with LGK974 in all cell lines. There was no difference in effectiveness between cell lines resistant or sensitive to TMZ. Interestingly, following TMZ treatment or irradiation cells showed slightly decreased canonical WNT pathway activity. Furthermore, non-canonical target genes and receptors were upregulated under chemo- and radiotherapy, suggesting a reciprocal activation of the non-canonical branch of WNT signaling pathway. This phenomenon might possibly be responsible for the development of resistance against standard chemo- and radiotherapy. Conclusions: We propose that GSCs undergo a shift from canonical to non-canonical WNT signaling to become treatment-resistant under standard chemo- and radiotherapy. Treatment with LGK974 results in therapeutic synergy at least in part via suppressing the therapy-induced activation of non-canonical WNT signaling. Our results confirm the potential benefit of combining TMZ or irradiation with the WNT inhibitor LGK974 to overcome therapy-resistance in GBM. Citation Format: Abigail K. Suwala, Ulf D. Kahlert, Jaroslaw Maciaczyk. Pharmacological WNT-inhibition acts synergistically with chemo- and radiotherapy by overcoming treatment-resistance in glioma stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2515.

Abstract 1699: Protumoral role of trans fatty acid in colorectal cancer

Abstract Trans fatty acids (TFAs) is a recent focus of health problems. TFA is a definitive risk factor for cardiovascular diseases and the death. TFA is also possible risk factor for Alzheimer's disease, obesity, diabetes, fatty liver, and ovulation infertility. Relation of TFA with carcinogenic risk is controversy; however, TFA is reported to increase the risk of breast cancer and prostate cancer. Elaidic acid (EA), a trans form of oleic acid, enhances cancer cell growth, invasion, and anti-apoptotic survival. By animal models, EA promotes tumor growth and metastasis to the lung, liver, and peritoneum. EA induces stemness in cancer cells through transactivation of EFGR via SRC from GPR40/120 as receptors in EA-integrated cholesterol raft. Activated EGFR relays the signals to activate canonical and non-canonical wnt pathway and to inactivate notch1 pathways. EA also increases miR-494, which inhibits cell differentiation through decrease of target genes. Continuous EA feeding with dosage alteration increased cancer cell stemness. EA diminishes the efficiency of 5-fluorouracil by increase of residual cancer stem cells. These findings suggest that TFA is a relevant cancer promoting factor. Decision of removal of TFA from foods by FDA might provide an impact to cancer clinics. Citation Format: Hitoshi Ohmori, Toshio Kuwai, Yasuhiko Kitadai, Kiyomu Fujii, Rina Fujiwara, Yukiko Nishiguchi, Tomonori Sasahira, Hiroki Kuniyasu. Protumoral role of trans fatty acid in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1699.

Abstract CT146: A phase IB study of the combination of AZD6244 hydrogen sulfate (selumetinib) and cyclosporin A (CsA) in patients with advanced solid tumors with an expansion cohort in metastatic colorectal cancer (mCRC)

Abstract Background: Targeting MEK is of great interest in the development of novel agents for treatment of a variety of malignancies. Resistance to MEK inhibitors has delayed the development of novel agents, and better strategies are needed to overcome acquired resistance. Preclinical studies performed at the University of Colorado in KRAS mutant cell lines have shown that several members of the Wnt pathway are overexpressed in cell lines resistant to the MEK inhibitor, selumetinib. Gene set enrichment analysis and synthetic lethal screens demonstrated that genes involved in the canonical and non-canonical Wnt pathways were upregulated in selumetinib-resistant CRC cell lines, whereas the combination of selumetinib and cyclosporin A (CsA), a WNT pathway modulator, demonstrated antitumor activity in patient-derived xenograft (PDX) models. We are conducting an NCI CTEP-approved Phase I/IB trial of the combination of selumetinib and CsA. Biomarkers of response to therapy are being co-developed in a preclinical trial of MEK/WNT inhibition in PDX models. We hypothesize that this combination will be safe, potentially effective in patients with mCRC and that upregulation of FZD2 may predict for sensitivity. Methods: This is a dose-escalation phase I trial investigating the combination of selumetinib and CsA in patients with solid tumors with an expansion cohort in patients with mCRC (n = 20). The expansion cohort will utilize a “run-in” of MEK inhibition alone to evaluate adaptive mechanisms of resistance that may identify those patients most likely to respond to the combination. An adaptive statistical design will be used to assess both KRAS/NRAS wild-type and mutant CRC to determine whether either or both subsets should be studied further in phase II trials. Results: As of January 15, 2016, a total of 18 patients have been enrolled and treated with selumetinib and CsA in the escalation phase. One DLT was reported in cohort 1 (G3 hypertension), and three DLTs (G3 hypertension, rash and elevated creatinine) were reported in cohort 2 (n = 12). Grade 1 or 2 nausea (67%) and rash (50%) were reported as the most common AEs. One unconfirmed partial response was noted (CRC KRAS mut), and 9 patients exhibited stable disease as their best response. One patient maintained stable disease for over 9 months. Disease control rate was 56% in the escalation cohort. Preclinical studies are currently being performed using next-generation MEK and WNT inhibitors in CRC PDX models. Conclusions: The combination of selumetinib and CsA is well tolerated with evidence of preliminary anti-tumor activity. Selumetinib at 75mg/kg BID and CsA at 2mg/kg was selected as the recommended phase 2 dose. The dose expansion in patients with metastatic CRC is underway. Preclinical studies and tissue acquisition for correlative studies are ongoing. Citation Format: Anna Capasso, Arvind Dasari, A. Craig Lockhart, Mark Stein, Hanna Sanoff, James Lee, Aaron Hansen, Tanios Bekaii-Saab, Sarah Rippke, James Yao, Funda Meric-Bernstam, S Gail Eckhardt, Christopher h. Lieu. A phase IB study of the combination of AZD6244 hydrogen sulfate (selumetinib) and cyclosporin A (CsA) in patients with advanced solid tumors with an expansion cohort in metastatic colorectal cancer (mCRC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT146.

Abstract 3354: Characterization of lung tumorspheres by gene expression and flow cytometry: differential expression in CSC-related markers and signaling pathways

Abstract Chemoresistance, progression and metastasis have made of lung cancer the first cause of cancer mortality. These features were linked to a subpopulation of cells, named cancer stem cells (CSCs), which remain largely unknown. The aim of this study was to isolate and characterize CSCs from lung cancer cell-lines and tumor-tissue from resectable non-small cell lung cancer (NSCLC). Methods: Tumor cells from resected NSCLC and cell lines (H1650, H1993, A549, and PC9) were grown in monolayer and as spheroids. RTqPCR was performed to analyze the mRNA expression of CSCs-related genes: CSC-markers (EPCAM1, ALDH1A1, CD166, ABCG2, CD44, CD133); pluripotency genes (KLF4, OCT4, NANOG, SOX2, MYC, CCND1); Notch pathway (NOTCH1, NOTCH3, HEY1); Wnt pathway (WNT1, WNT5A, DKK1, FZD7) and Hedgehog pathway (SMO, PTCH1, SHH, GLI1). ACTB and CDKN1B were used as endogenous controls for relative expression calculation. The expression of lung stem cell markers EpCAM, CD166, E-cadherin, CD90, CD44, CD34, CD133 and ABCG2 was assessed by flow cytometry. The tumor-initiating cell capacity of selected lung-spheres was tested in vivo to confirm tumorigenicity. Results: Lung-tumorspheres had increased expression of EPCAM, CD44 and ALDH1A1 (p = 0.028, p = 0.021 and p = 0.043, respectively) when compared to cells grown in adherence. Likewise, NANOG, KLF4 and OCT4 tended to be more expressed in tumorspheres. Relative gene expression of NOTCH1 was also higher in spheroids than in monolayer cells (p = 0.028), in concordance with the same tendency observed in NOTCH3. Similarly, RTqPCR analysis revealed a possible activation of the canonical Wnt pathway in tumorspheres, with high expression levels of the downstream effector gene CCND1 (p = 0.05), along with a repression of DKK1 inhibitor. Regarding to the non-canonical Wnt pathway, its activator WNT5A showed lower expression levels in spheroids compared to monolayer-culture cells. Concerning the expression levels of Hedgehog pathway's genes, we found that SMO and PTCH1 were underexpressed in lung-tumorspheres compared with their paired adherent-cultured cells (p = 0.028 and p = 0.069). Flow cytometry revealed that EpCAM and CD44 were highly expressed in lungspheres obtained from cell lines and primary tumors. The expression of CD166 differed among the cell lines. Furthermore, EpCAM+/CD90- subpopulation were the ones able to induce tumor in xenotrasplant mouse model demostrating tumor-initiating capacity in vivo. Conclusions: Lung-tumorspheres derived from cancer cell lines and primary tumor tissues show increased levels of EpCAM and others CSC markers. Genes related to Notch and Wnt signaling pathways were more expressed in spheroids compared to the cells grown in adherence, suggesting both pathways as interesting lung-CSC targets. Supported by grants RD12/0036/0025 from RTICC, PI12-02838 and PI12-0956 from ISCIII and SEOM/2012.[E.M.-M., A.H.-P. and A.M.-R. contributed equally to this work.] Citation Format: Ester Munera-Maravilla, Alejandro Herreros-Pomares, Alicia Martínez-Romero, Sandra Tejedor, Silvia Calabuig-Fariñas, Eloísa Jantus-Lewintre, Rut Lucas, Eva Escorihuela, Rosa Farràs, Carlos Camps. Characterization of lung tumorspheres by gene expression and flow cytometry: differential expression in CSC-related markers and signaling pathways. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3354.

Can Wnt5a and Wnt non-canonical pathways really mediate adipocyte de-differentiation in a tumour microenvironment?

Wnt5a has been recently reported as a possible triggering factor of adipocyte de-differentiation into an adipocyte-derived fibroblast in the tumour microenvironment of pancreas cancer. The Wnt/β-catenin pathway was described in processes involving de-differentiation and epithelial-mesenchymal transition but some Wnt family-belonging molecules exert an adipogenic role on adipocyte, while other ones, such as Wnt10b or Wnt3a, an anti-adipogenic role. Although this ability depends on the different tumoural microenvironments, it is intriguing to ascertain if some Wnt molecules, participating in the non-canonical pathway, may be targeted as fundamental factors able to trigger the desmoplastic reaction of peritumoural white adipose tissue.

Aberrant DNA methylation of WNT pathway genes in the development and progression of CIMP-negative colorectal cancer

ABSTRACTThe WNT signaling pathway has an essential role in colorectal carcinogenesis and progression, which involves a cascade of genetic and epigenetic changes. We aimed to analyze DNA methylation affecting the WNT pathway genes in colorectal carcinogenesis in promoter and gene body regions using whole methylome analysis in 9 colorectal cancer, 15 adenoma, and 6 normal tumor adjacent tissue (NAT) samples by methyl capture sequencing. Functional methylation was confirmed on 5-aza-2′-deoxycytidine-treated colorectal cancer cell line datasets. In parallel with the DNA methylation analysis, mutations of WNT pathway genes (APC, β-catenin/CTNNB1) were analyzed by 454 sequencing on GS Junior platform. Most differentially methylated CpG sites were localized in gene body regions (95% of WNT pathway genes). In the promoter regions, 33 of the 160 analyzed WNT pathway genes were differentially methylated in colorectal cancer vs. normal, including hypermethylated AXIN2, CHP1, PRICKLE1, SFRP1, SFRP2, SOX17, and hypomethylated CACYBP, CTNNB1, MYC; 44 genes in adenoma vs. NAT; and 41 genes in colorectal cancer vs. adenoma comparisons. Hypermethylation of AXIN2, DKK1, VANGL1, and WNT5A gene promoters was higher, while those of SOX17, PRICKLE1, DAAM2, and MYC was lower in colon carcinoma compared to adenoma. Inverse correlation between expression and methylation was confirmed in 23 genes, including APC, CHP1, PRICKLE1, PSEN1, and SFRP1. Differential methylation affected both canonical and noncanonical WNT pathway genes in colorectal normal-adenoma-carcinoma sequence. Aberrant DNA methylation appears already in adenomas as an early event of colorectal carcinogenesis.

Secreted Frizzled-related Protein 2 (sFRP2) Redirects Non-canonical Wnt Signaling from Fz7 to Ror2 during Vertebrate Gastrulation

Convergent extension movements during vertebrate gastrulation require a balanced activity of non-canonical Wnt signaling pathways, but the factors regulating this interplay on the molecular level are poorly characterized. Here we show that sFRP2, a member of the secreted frizzled-related protein (sFRP) family, is required for morphogenesis and papc expression during Xenopus gastrulation. We further provide evidence that sFRP2 redirects non-canonical Wnt signaling from Frizzled 7 (Fz7) to the receptor tyrosine kinase-like orphan receptor 2 (Ror2). During this process, sFRP2 promotes Ror2 signal transduction by stabilizing Wnt5a-Ror2 complexes at the membrane, whereas it inhibits Fz7 signaling, probably by blocking Fz7 receptor endocytosis. The cysteine-rich domain of sFRP2 is sufficient for Ror2 activation, and related sFRPs can substitute for this function. Notably, direct interaction of the two receptors via their cysteine-rich domains also promotes Ror2-mediated papc expression but inhibits Fz7 signaling. We propose that sFRPs can act as a molecular switch, channeling the signal input for different non-canonical Wnt pathways during vertebrate gastrulation.

Current results of a phase I study of DKN-01, an anti-DKK1 antibody, in combination with paclitaxel (P) in patients (pts) with advanced DKK1+ esophageal cancer (EC) or gastro-esophageal junction tumors (GEJ).

e15525Background: DKK1 is an indirect inhibitor of canonical Wnt signaling and has been shown to directly activate non-canonical Wnt pathways including Rho, ROCK and JNK. High DKK1 tissue concentra...

Wnt, Frizzled, and sFRP gene expression patterns during gastrulation in the starfish Patiria (Asterina) pectinifera

By the initial phase of gastrulation, Wnt pathway regulation mediates endomesoderm specification and establishes the animal-vegetal axis, thereby leading to proper gastrulation in starfish. To provide insight into the ancestral mechanism regulating deuterostome gastrulation, we identified the gene expression patterns of Wnt, Frizzled (Fz), and secreted frizzled-related protein (sFRP) family genes, which play a role in the initial stage of the Wnt pathway, in starfish Patiria (Asterina) pectinifera embryos using whole mount in situ hybridization. We identified ten Wnt, four Fz, and two sFRP paralogues. From the hatching blastula to the late gastrula stage, the majority of the Wnt genes and both Fz5/8 and sFRP1/5 were expressed in the posterior and anterior half of the embryo, respectively. Wnt8, Fz1, and Fz4 showed restricted expression in the lateral ectoderm. On the other hand, several genes were expressed de novo in the restricted domain of the archenteron at the late gastrula stage. These results suggest that the canonical and/or non-canonical Wnt pathway might implicate endomesoderm specification, anterior-posterior axis establishment, anterior-posterior patterning, and archenteron morphogenesis in the developmental context of starfish embryos. From comparison with the expression patterns observed in Patria miniata, we consider that the Wnt pathway is conserved among starfishes.

Epithelia-derived wingless regulates dendrite directional growth of drosophila ddaE neuron through the Fz-Fmi-Dsh-Rac1 pathway.

BackgroundProper dendrite patterning is critical for the receiving and processing of information in the nervous system. Cell-autonomous molecules have been extensively studied in dendrite morphogenesis; however, the regulatory mechanisms of environmental factors in dendrite growth remain to be elucidated.ResultsBy evaluating the angle between two primary dendrites (PD-Angle), we found that the directional growth of the primary dendrites of a Drosophila periphery sensory neuron ddaE is regulated by the morphogen molecule Wingless (Wg). During the early stage of dendrite growth, Wg is expressed in a group of epithelial cells posteriorly adjacent to ddaE. When Wg expression is reduced or shifted anteriorly, the PD-Angle is markedly decreased. Furthermore, Wg receptor Frizzled functions together with Flamingo and Dishevelled in transducing the Wg signal into ddaE neuron, and the downstream signal is mediated by non-canonical Wnt pathway through Rac1.ConclusionsIn conclusion, we reveal that epithelia-derived Wg plays a repulsive role in regulating the directional growth of dendrites through the non-canonical Wnt pathway. Thus, our findings provide strong in vivo evidence on how environmental signals serve as spatial cues for dendrite patterning.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-016-0228-0) contains supplementary material, which is available to authorized users.

Wnt9A Induction Linked to Suppression of Human Colorectal Cancer Cell Proliferation.

Most studies of Wnt signaling in malignant tissues have focused on the canonical Wnt pathway (CWP) due to its role in stimulating cellular proliferation. The role of the non-canonical Wnt pathway (NCWP) in tissues with dysregulated Wnt signaling is not fully understood. Understanding NCWP’s role is important since these opposing pathways act in concert to maintain homeostasis in healthy tissues. Our preliminary studies demonstrated that LiCl inhibited proliferation of primary cells derived from colorectal cancer (CRC). Since LiCl stimulates cell proliferation in normal tissues and NCWP suppresses it, the present study was designed to investigate the impact of NCWP components in LiCl-mediated effects. LiCl-mediated inhibition of CRC cell proliferation (p < 0.001) and increased apoptosis (p < 0.01) coincided with 23-fold increase (p < 0.025) in the expression of the NCWP ligand, Wnt9A. LiCl also suppressed β-catenin mRNA (p < 0.03), total β-catenin protein (p < 0.025) and the active form of β-catenin. LiCl-mediated inhibition of CRC cell proliferation was partially reversed by IWP-2, and Wnt9A antibody. Recombinant Wnt9A protein emulated LiCl effects by suppressing β-catenin protein (p < 0.001), inhibiting proliferation (p < 0.001) and increasing apoptosis (p < 0.03). This is the first study to demonstrate induction of a NCWP ligand, Wnt9A as part of a mechanism for LiCl-mediated suppression of CRC cell proliferation.

PPARγ; and Wnt Signaling in Adipogenic and Osteogenic Differentiation of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) arise from a variety of tissues, including bone marrow and adipose tissue and, accordingly, have the potential to differentiate into multiple cell types, including osteoblasts and adipocytes. Research on MSCs to date has demonstrated that a large number of transcription factors and ectocytic or intrastitial signaling pathways regulate adipogenic and osteogenic differentiation. A theoretical inverse relationship exists in adipogenic and osteogenic lineage commitment and differentiation, such that signaling pathways induce adipogenesis at the expense of osteogenesis and vice versa. For example, peroxisome proliferator-activated receptor γ(PPARγ), which belongs to the nuclear hormone receptor superfamily of ligand-activated transcription factors, is known to function as a master transcriptional regulator of adipocyte differentiation, and inhibit osteoblast differentiation. Moreover, recent studies have demonstrated that inducers of osteogenic differentiation, such as bone morphogenetic protein (BMP) and Wnt, inhibit the function of PPARγ transactivation during MSC differentiation towards adipocytes through a variety of mechanisms. To illustrate this, the canonical Wnt/β-catenin pathway represses expression of PPARγ mRNA, whereas the noncanonical Wnt pathway activates histone methyltransferases that inhibit PPARγ transactivation via histone H3 lysine 9 (H3K9) methylation of its target genes. The role of microRNAs (miRNAs) in adipogenesis and osteoblastogenesis is garnering increased attention, and studies in this area have shed light on the integration of miRNAs with Wnt signaling and transcription factors such as Runx2 and PPARγ. This review summarizes our current understanding of the mechanistic basis of these signaling pathways, and indicates future clinical applications for stem cell-based cell transplantation and regenerative therapy.

Combination of melatonin and Wnt-4 promotes neural cell differentiation in bovine amniotic epithelial cells and recovery from spinal cord injury.

Although melatonin has been shown to exhibit a wide variety of biological functions, its effects on promoting differentiation of neural cells remain unknown. Wnt signaling mediates major developmental processes during embryogenesis and regulates maintenance, self-renewal, and differentiation of adult mammalian stem cells. However, the role of the noncanonical Wnt pathway during neurogenesis remains poorly understood. In this study, the amniotic epithelial cells ( AECs) were isolated from bovine amnion and incubated with various melatonin concentrations (0.01, 0.1, 1, 10, or 100 μm) and 5 × 10(-5) m all-trans retinoic acid (RA) for screening optimum culture medium of neural differentiation, compared with each groups, 1 μm melatonin and 5 × 10(-5) m RA were selected to induce neural differentiation of AECs, and then siMT1, siMT2, oWnt-4, and siWnt-4 were expressed in AECs to research role of these genes in neural differentiation. Efficiency of neural differentiation was evaluated after expressed above genes using flow cytometry. Cell function of neural cells was demonstrated in vivo using spinal cord injury model after cell transplantation, and damage repair of spinal cord was assessed using cell tracking and Basso, Beattie, Bresnahan Locomotor Rating Scale scores. Results demonstrated that melatonin stimulated melatonin receptor 1, which subsequently increased bovine amniotic epithelial cell vitality and promoted differentiation into neural cells. This took place through cooperation with Wnt-4. Additionally, following cotreatment with melatonin and Wnt-4, neurogenesis gene expression was significantly altered. Furthermore, single inhibition of melatonin receptor 1 or Wnt-4 expression decreased expression of neurogenesis-related genes, and bovine amniotic epithelial cell-derived neural cells were successfully colonized into injured spinal cord, which suggested participation in tissue repair.

The effect of Wnt9A induced by lithium chloride on β-catenin and colorectal cancer cell proliferation.

631 Background: Malignant tissues are characterized by uncontrolled proliferation and dysfunctional differentiation which often results from dysregulated Wnt signaling. β-catenin, a second messenger upregulated in neoplastic tissues is utilized by the canonical Wnt pathway (CWP) to promote cellular proliferation. In healthy tissues CWP is stringently controlled by components of the non-canonical Wnt pathway (NCWP). The NCWP, which does not utilize β-catenin for its activity, acts in concert with the CWP to maintain homeostasis in healthy tissues. Our recent studies in CRC cells indicate that LiCl increases expression of the NCWP ligand, Wnt9A leading to activation of the NCWP. Suppressing Wnt9A significantly attenuates LiCl-mediated inhibition of CRC cell proliferation. The present study investigated the capacity of Wnt9A to directly modulate expression of β-catenin mRNA and/or protein levels in CRC cells, and the effects on proliferation and apoptosis. Methods: Primary CRC lines established from resected tumors of 5 patients with metastatic disease were treated with LiCl and Wnt9A protein. Cellular proliferation and apoptosis were determined by MTS and caspase 3 assays. Total and activated β-catenin protein were determined by ELISA and Wnt pathway genes and β-catenin mRNA were determined with PCR arrays. Results: LiCl inhibited CRC proliferation (p&lt;0.001) and increased apoptosis (p&lt;0.01) which coincided with a 23-fold (p&lt;0.025) increase of the NCWP ligand Wnt9A. LiCl decreased β-catenin mRNA (p&lt;0.03) and suppressed protein levels of the total (p&lt;0.025) and the activated form of β-catenin. Recombinant Wnt9A protein emulated LiCl effects by inhibiting proliferation (p&lt;0.001), increasing apoptosis (p&lt;0.03) and suppressing activated β-catenin protein (p&lt;0.001) in CRC cells. Conclusions: This study demonstrates a novel mechanism for inhibiting CRC cell proliferation through suppression of activated β-catenin levels by the non-canonical Wnt ligand, Wnt9A. These results suggest that in CRC tumors that are driven by β-catenin, activation of the non-canonical pathway and/or direct induction of or treatment with Wnt9A protein may be therapeutically beneficial.

Take the Wnt out of the inflammatory sails: modulatory effects of Wnt in airway diseases

Bronchial asthma and chronic obstructive pulmonary disease (COPD) are chronic diseases that are associated with inflammation and structural changes in the airways and lungs. Recent findings have implicated Wnt pathways in critically regulating inflammatory responses, especially in asthma. Furthermore, canonical and noncanonical Wnt pathways are involved in structural changes such as airway remodeling, goblet cell metaplasia, and airway smooth muscle (ASM) proliferation. In COPD, Wnt pathways are not only associated with structural changes in the airways but also involved in the development of emphysema. The present review summarizes the role and function of the canonical and noncanonical Wnt pathway with regard to airway inflammation and structural changes in asthma and COPD. Further identification of the role and function of different Wnt molecules and pathways could help to develop novel therapeutic options for these diseases.

Abstract IA08: Developmental insights into breast cancer intratumoral heterogeneity

Abstract Breast cancer is no longer considered a single disease, but instead is made up of multiple subtypes with genetically and epigenetically heterogeneous tumors composed of numerous clones. Both the hierarchical cancer stem cell and clonal evolution models have been invoked to help explain this intratumoral heterogeneity. Several recent studies have helped define the functional interactions among the different cellular subpopulations necessary for the evolution of this complex ecosystem. These interactions involve paracrine interactions that include locally acting Wnt family members, reminiscent of the signaling pathways important for normal mammary gland development and stem cell self-renewal. For example, mammary gland stem cell self-renewal is regulated by paracrine signaling involving Wnt4 and RSPO1 secreted by differentiated mammary luminal cells under the influence of systemic steroid hormones. Furthermore, interactions between non-canonical and canonical Wnt pathways play an important role in mammary gland development and cell fate determination [1]. Using a genetically engineered p53 null triple negative breast cancer model, we described a novel feedback loop in which the breast cancer stem cells(CSC) function through the action of paracrine mediators secreted by the CSC progeny[2], similar to what is observed in the normal mammary gland. We demonstrated the existence of cross-talk between tumor cells via Wnt2, Cxcl12 and IL6 signaling, indicating the non-cell-autonomous properties and importance of cooperativity between tumor subpopulations in tumor initiation and progression. Ligands expressed in the mesenchymal progeny cells produced through the asymmetric division of CSCs interacted with receptors on the CSCs. Knockdown of the ligands in the mesenchymal cells and their respective receptors in the CSCs resulted in decreased mammosphere formation in co-cultures. Furthermore, knockdown of Wnt 2 in the mesenchymal cells resulted in increased tumor latency when limiting numbers of CSCs were transplanted into the cleared mammary fat pad. The functional interactions between CSCs and mesenchymal population of cells may provide an improved niche microenvironment, especially for those tumors initiated from extremely low numbers of the CSCs. These studies indicate that similar regulatory mechanisms may exist for the maintenance of both normal and tumor stem cells with respect to their symmetric and asymmetric division. A better understanding of these interactions, especially in the metastatic setting, will be important for the development of improved combinatorial therapies designed to prevent relapse and to ultimately decrease mortality.

Phase I study of DKN-01, an anti-DKK1 antibody, in combination with paclitaxel (pac) in patients (pts) with DKK1+ relapsed or refractory esophageal cancer (EC) or gastro-esophageal junction tumors (GEJ).

111 Background: DKK1, a secreted protein, is an indirect inhibitor of canonical Wnt signaling and was recently shown to directly activate non-canonical Wnt pathways including Rho, ROCK and JNK. High DKK1 concentrations in EC tumor tissue correlate with shorter overall survival (OS); published data suggests increased activity of non-canonical signaling by DKK1 may be a reason for shortened OS. DKN-01 is a humanized monoclonal antibody to DKK1 with demonstrated activity in non-small cell lung cancer. This study evaluated the MTD, safety and early activity of DKN-01 in combination with pac in EC and GEJ cancer pts. Methods: In Part A, pts with DKK1+ EC or GEJ cancer by IHC staining received DKN-01 in two dose escalation cohorts (150 or 300 mg) with pac 80 mg/m2 on days 1, 8, 15 and 22 of each 28 day cycle. Part B (dose expansion) is ongoing. Responses were assessed using RECIST version 1.1. Results: &gt; 90% of screened EC (70 pts) had DKK1+ tumor tissue. In Part A, 9 pts with advanced EC or GEJ cancer were enrolled: 3 in Cohort 1 (150 mg) and 6 in Cohort 2 (300 mg); 5 pts had a diagnosis of EC [3 with adenocarcinoma (AC); 2 with squamous cell] and 4 pts with GEJ AC. Median number of prior therapies was 2 (range 1, 8). One DLT, G3 peripheral neuropathy, was seen at 300 mg. Frequent adverse events (AEs) were cough (5 pts), alopecia, leukopenia, neutropenia and peripheral/sensory neuropathy (each 4 pts) and fatigue, anemia, and nausea (each 3 pts); AEs were mostly Grade 1/2. The MTD of DKN-01 + pac was determined to be 300 mg. Among the 7 AC pts, 3 pts had a partial response (one having had prior pac); 3 pts had stable disease. Responders were on therapy between 3 and 10 mos. Conclusions: Part A is complete. Most EC and GEJ pts express tumor DKK1 and have obviated the need for evaluation of tumor DKK1+ expression as a study entry prerequisite. Combination of DKN-01+ pac is well-tolerated; the addition of DKN-01 to pac does not appear to alter the known pac safety profile. There are initial signs of enhanced clinical activity with DKN-01; further investigation is ongoing. Clinical trial information: NCT02013154. Clinical trial information: NCT02013154.

Dkk3 prevents familial dilated cardiomyopathy development through Wnt pathway

To date, the role of Dickkopf 3 (Dkk3) on the pathogenesis of familial dilated cardiomyopathy (FDCM), and whether and how Dkk3 interferes with Wnt signaling in heart tissues remains unknown. Here, we demonstrate that strong Dkk3 expression was markedly downregulated in adult hearts from WT mice, and Dkk3 expression was upregulated suddenly in hearts from DCM mouse models. Using Dkk3 transgenic and knockout mice, as well as cTnTR141W transgenic mice, which manifests progressive chamber dilation and contractile dysfunction and has pathologic phenotypes similar to human DCM patients, we determined that transgenic expression of Dkk3 increased survival rate, improved cardiac morphology breakage and dysfunction, and ameliorated cardiac pathological changes in the cTnTR141W mice. In contrast, Dkk3 knockout reduced the survival rate and aggravated the pathological phenotypes of the cTnTR141W mice. The protective effects of Dkk3 appeared clearly at 3 months of age, peaked at 6 months of age, and decreased at 10 months of age in the cTnTR141W mice. Furthermore, we determined that Dkk3 upregulated Dvl1 (Dishevelled 1) and key proteins of the canonical Wnt pathway (cytoplasmic and nuclear β-catenin, c-Myc, and Axin2) and downregulated key proteins of the noncanonical Wnt pathway (c-Jun N-terminal kinase (JNK), Ca2+/calmodulin-dependent protein kinase II (CAMKII), and histone deacetylase 4 (HDAC4)). In contrast, Dkk3 knockout reversed these changes in the cTnTR141W mice. In summary, Dkk3 could prevent FDCM development in mice, especially in the compensatory stage, and probably through activation of the canonical and inhibition of the noncanonical Wnt pathway, which suggested that Dkk3 could serve as a therapeutic target for the treatment of cardiomyopathy and heart failure.

GEP analysis validates high risk MDS and acute myeloid leukemia post MDS mice models and highlights novel dysregulated pathways.

BackgroundIn spite of the recent discovery of genetic mutations in most myelodysplasic (MDS) patients, the pathophysiology of these disorders still remains poorly understood, and only few in vivo models are available to help unravel the disease.MethodsWe performed global specific gene expression profiling and functional pathway analysis in purified Sca1+ cells of two MDS transgenic mouse models that mimic human high-risk MDS (HR-MDS) and acute myeloid leukemia (AML) post MDS, with NRASD12 and BCL2 transgenes under the control of different promoters MRP8NRASD12/tethBCL-2 or MRP8[NRASD12/hBCL-2], respectively.ResultsAnalysis of dysregulated genes that were unique to the diseased HR-MDS and AML post MDS mice and not their founder mice pointed first to pathways that had previously been reported in MDS patients, including DNA replication/damage/repair, cell cycle, apoptosis, immune responses, and canonical Wnt pathways, further validating these models at the gene expression level. Interestingly, pathways not previously reported in MDS were discovered. These included dysregulated genes of noncanonical Wnt pathways and energy and lipid metabolisms. These dysregulated genes were not only confirmed in a different independent set of BM and spleen Sca1+ cells from the MDS mice but also in MDS CD34+ BM patient samples.ConclusionsThese two MDS models may thus provide useful preclinical models to target pathways previously identified in MDS patients and to unravel novel pathways highlighted by this study.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0235-8) contains supplementary material, which is available to authorized users.