Dr. Rob Winn

The variation of macrophage functions suggests the involvement of multiple signalling pathways in fine tuning their differentiation. Macrophages that originate from monocytes in the blood migrate to tissue in response to homeostatic or ‘danger’ signals and undergo substantial morphological and functional modifications to meet the needs of the dominant signals in the microenvironment. Wnts are secreted glycoproteins that play a significant role in organ and cell differentiation, yet their impact… Show more

The variation of macrophage functions suggests the involvement of multiple signalling pathways in fine tuning their differentiation. Macrophages that originate from monocytes in the blood migrate to tissue in response to homeostatic or ‘danger’ signals and undergo substantial morphological and functional modifications to meet the needs of the dominant signals in the microenvironment. Wnts are secreted glycoproteins that play a significant role in organ and cell differentiation, yet their impact on monocyte differentiation is not clear. In this study, we assessed the role of Wnt1 and Wnt7a on the differentiation of monocytes and the subsequent phenotype and function of monocyte‐derived macrophages (MDMs). We show that Wnt7a decreased the expression of CD14, CD11b, CD163 and CD206, whereas Wnt1 had no effect. The Wnt7a effect on CD11b was also observed in the brain and spleen of Wnt7a−/− adult brain mouse tissue and in embryonic Wnt7a−/− tissue. Wnt7a reduced the phagocytic capacity of M‐MDMs, decreased interleukin‐10 (IL‐10) and IL‐12 secretion and increased IL‐6 secretion. Collectively, these findings demonstrate that Wnt7a generates an MDM phenotype with both pro‐inflammatory and alternative MDM cytokine profiles and reduced phagocytic capacity. As such, Wnt7a can have a significant impact on macrophage responses in health and disease. Show less

Atypical protein kinase C ζ (PKCζ) forms an apico-basal polarity complex with Partitioning Defective (Pard) 3 and Pard6 to regulate normal epithelial cell apico-basolateral polarization. The dissociation of the PKCζ/Pard3/Pard6 complex is essential for the disassembly of the tight/adherens junction and epithelial-mesenchymal transition (EMT) that is critical for tumor spreading. Loss of cell polarity and epithelial organization is strongly correlated with malignancy and tumor progression in… Show more

Atypical protein kinase C ζ (PKCζ) forms an apico-basal polarity complex with Partitioning Defective (Pard) 3 and Pard6 to regulate normal epithelial cell apico-basolateral polarization. The dissociation of the PKCζ/Pard3/Pard6 complex is essential for the disassembly of the tight/adherens junction and epithelial-mesenchymal transition (EMT) that is critical for tumor spreading. Loss of cell polarity and epithelial organization is strongly correlated with malignancy and tumor progression in some other cancer types. However, it is unclear whether the PKCζ/Pard3/Pard6 complex plays a role in the progression of non-small-cell lung cancer (NSCLC). We found that hypoxia downregulated the PKCζ/Pard3/Pard6 complex, correlating with induction of lung cancer cell migration and invasion. Silencing of the PKCζ/Pard3/Pard6 polarity complex components induced lung cancer cell EMT, invasion, and colonization in vivo. Suppression of Pard3 was associated with altered expression of genes regulating wound healing, cell apoptosis/death and cell motility, and particularly upregulation of MAP3K1 and fibronectin which are known to contribute to lung cancer progression. Human lung adenocarcinoma tissues expressed less Pard6b and PKCζ than the adjacent normal tissues and in experimental mouse lung adenocarcinoma, the levels of Pard3 and PKCζ were also decreased. In addition, we showed that a methylation locus in the gene body of Pard3 is positively associated with the expression of Pard3 and that methylation of the Pard3 gene increased cellular sensitivity to carboplatin, a common chemotherapy drug. Suppression of Pard3 increased chemoresistance in lung cancer cells. Together, these results suggest that reduced expression of PKCζ/Pard3/Pard6 contributes to NSCLC EMT, invasion, and chemoresistance. Show less

AU-rich element-binding proteins (ARE-BPs) offer post-transcriptional regulation of gene expression via physical interaction and recruitment of RNA decay machinery to the AU-rich elements within the 3′-UTR of the target transcripts. However, the role of ARE-BPs in lung cancer remains poorly understood. In this study, we have identified that K-homology splicing regulatory protein (KSRP), an ARE-BP, is robustly up-regulated in human lung cancer. Importantly, Kaplan-Meier survival analysis… Show more

AU-rich element-binding proteins (ARE-BPs) offer post-transcriptional regulation of gene expression via physical interaction and recruitment of RNA decay machinery to the AU-rich elements within the 3′-UTR of the target transcripts. However, the role of ARE-BPs in lung cancer remains poorly understood. In this study, we have identified that K-homology splicing regulatory protein (KSRP), an ARE-BP, is robustly up-regulated in human lung cancer. Importantly, Kaplan-Meier survival analysis indicated that elevated KSRP expression was correlated with poor overall survival of lung cancer patients. Furthermore, cigarette smoke, a leading risk factor for lung cancer, was also identified to be an important contributor to increased KSRP expression. Remarkably, silencing of KSRP decreased cell proliferation, reversed anchorage-independent growth, and reduced migration/invasion, suggesting an oncogenic role for KSRP in lung cancer. Finally, we provide mechanistic evidence that KSRP promotes the down-regulation of Spry4 by a previously unidentified mechanism, i.e. post-transcriptional mRNA regulation. Show less

Controlled distribution of lipids across various cell membranes is crucial for cell homeostasis and regulation. We developed an imaging method that allows simultaneous in situ quantification of cholesterol in two leaflets of the plasma membrane (PM) using tunable orthogonal cholesterol sensors. Our imaging revealed marked transbilayer asymmetry of PM cholesterol (TAPMC) in various mammalian cells, with the concentration in the inner leaflet (IPM) being 1/412-fold lower than that in the outer… Show more

Controlled distribution of lipids across various cell membranes is crucial for cell homeostasis and regulation. We developed an imaging method that allows simultaneous in situ quantification of cholesterol in two leaflets of the plasma membrane (PM) using tunable orthogonal cholesterol sensors. Our imaging revealed marked transbilayer asymmetry of PM cholesterol (TAPMC) in various mammalian cells, with the concentration in the inner leaflet (IPM) being 1/412-fold lower than that in the outer leaflet (OPM). The asymmetry was maintained by active transport of cholesterol from IPM to OPM and its chemical retention at OPM. Furthermore, the increase in the IPM cholesterol level was triggered in a stimulus-specific manner, allowing cholesterol to serve as a signaling lipid. We found excellent correlation between the IPM cholesterol level and cellular Wnt signaling activity, suggesting that TAPMC and stimulus-induced PM cholesterol redistribution are crucial for tight regulation of cellular processes under physiological conditions. Show less

Cellular senescence is an initial barrier for carcinogenesis. However, the signaling mechanisms that trigger cellular senescence are incompletely understood, particularly in vivo. Here we identify Wnt7a as a novel upstream inducer of cellular senescence. In two different mouse strains (C57Bl/6J and FVB/NJ), we show that the loss of Wnt7a is a major contributing factor for increased lung tumorigenesis owing to reduced cellular senescence, and not reduced apoptosis, or autophagy. Wnt7a-null mice… Show more

Cellular senescence is an initial barrier for carcinogenesis. However, the signaling mechanisms that trigger cellular senescence are incompletely understood, particularly in vivo. Here we identify Wnt7a as a novel upstream inducer of cellular senescence. In two different mouse strains (C57Bl/6J and FVB/NJ), we show that the loss of Wnt7a is a major contributing factor for increased lung tumorigenesis owing to reduced cellular senescence, and not reduced apoptosis, or autophagy. Wnt7a-null mice under de novo conditions and in both the strains display E-cadherin-to-N-cadherin switch, reduced expression of cellular senescence markers and reduced expression of senescence-associated secretory phenotype, indicating a genetic predisposition of these mice to increased carcinogen-induced lung tumorigenesis. Interestingly, Wnt7a induced an alternate senescence pathway, which was independent of β-catenin, and distinct from that of classical oncogene-induced senescence mediated by the well-known p16INK4a and p19ARF pathways. Mechanistically, Wnt7a induced cellular senescence via inactivation of S-phase kinase-associated protein 2, an important alternate regulator of cellular senescence. Additionally, we identified Iloprost, a prostacyclin analog, which initiates downstream signaling cascades similar to that of Wnt7a, as a novel inducer of cellular senescence, presenting potential future clinical translational strategies. Thus pro-senescence therapies using either Wnt7a or its mimic, Iloprost, might represent a new class of therapeutic treatments for lung cancer. Show less

γ-catenin (Plakoglobin), a well-described structural protein functioning at the adherens junctions and desmosomes, was shown to be either lost or weakly expressed in non-small cell lung cancer (NSCLC) cells and tumor tissues. However, the tumor suppressive affects of γ-catenin were not fully understood. In this study, we have identified a novel role for the affects of γ-catenin on non-small cell lung cancer (NSCLC) cell migration. Expression of γ-catenin in NSCLC cells resulted in reduced cell… Show more

γ-catenin (Plakoglobin), a well-described structural protein functioning at the adherens junctions and desmosomes, was shown to be either lost or weakly expressed in non-small cell lung cancer (NSCLC) cells and tumor tissues. However, the tumor suppressive affects of γ-catenin were not fully understood. In this study, we have identified a novel role for the affects of γ-catenin on non-small cell lung cancer (NSCLC) cell migration. Expression of γ-catenin in NSCLC cells resulted in reduced cell migration as determined by both scratch assays and trans-well cell migration assays. Moreover, the affects of γ-catenin on cell migration were observed to be p53-dependent. Mechanistically, the anti-migratory effects seen via γ-catenin were driven by the expression of hepatocyte growth factor activator inhibitor Type I (HAI-1 or SPINT-1), an upstream inhibitor of the c-MET signaling pathway. Furthermore, the re-expression of γ-catenin sensitized NSCLC cells to c-MET inhibitor-mediated growth inhibition. Taken together, we identify γ-catenin as a novel regulator of HAI-1, which is a critical regulator of HGF/c-MET signaling. Therefore, targeting γ-catenin-mediated HAI-1 expression might be a useful strategy to sensitize NSCLC to c-MET inhibitors. Show less

Protein arginine methyl transferase 1 (PRMT1) was shown to be up-regulated in cancers and important for cancer cell proliferation. However, the role of PRMT1 in lung cancer progression and metastasis remains incompletely understood. In the present study, we show that PRMT1 is an important regulator of epithelial-mesenchymal transition (EMT), cancer cell migration, and invasion, which are essential processes during cancer progression, and metastasis. Additionally, we have identified Twist1, a… Show more

Protein arginine methyl transferase 1 (PRMT1) was shown to be up-regulated in cancers and important for cancer cell proliferation. However, the role of PRMT1 in lung cancer progression and metastasis remains incompletely understood. In the present study, we show that PRMT1 is an important regulator of epithelial-mesenchymal transition (EMT), cancer cell migration, and invasion, which are essential processes during cancer progression, and metastasis. Additionally, we have identified Twist1, a basic helix-loop-helix transcription factor and a well-known E-cadherin repressor, as a novel PRMT1 substrate. Taken together, we show that PRMT1 is a novel regulator of EMT and arginine 34 (Arg-34) methylation of Twist1 as a unique "methyl arginine mark" for active E-cadherin repression. Therefore, targeting PRMT1-mediated Twist1 methylation might represent a novel strategy for developing new anti-invasive/anti-metastatic drugs. Moreover, methylated Twist1 (Arg-34), as such, could also emerge as a potential important biomarker for lung cancer. Show less

Ewing Sarcoma is a biologically aggressive bone and soft tissue malignancy affecting children and young adults. Ewing Sarcoma pathogenesis is driven by EWS/Ets fusion oncoproteins, of which EWS/Fli1 is the most common. We have previously shown that microRNAs (miRs) regulated by EWS/Fli1 contribute to the pro-oncogenic program in Ewing Sarcoma. Here we show that miR-22, an EWS/Fli1-repressed miR, is inhibitory to Ewing Sarcoma clonogenic and anchorage-independent cell growth, even at modest… Show more

Ewing Sarcoma is a biologically aggressive bone and soft tissue malignancy affecting children and young adults. Ewing Sarcoma pathogenesis is driven by EWS/Ets fusion oncoproteins, of which EWS/Fli1 is the most common. We have previously shown that microRNAs (miRs) regulated by EWS/Fli1 contribute to the pro-oncogenic program in Ewing Sarcoma. Here we show that miR-22, an EWS/Fli1-repressed miR, is inhibitory to Ewing Sarcoma clonogenic and anchorage-independent cell growth, even at modest overexpression levels. Our studies further identify the H3K9me1/2 histone demethylase KDM3A (JMJD1A/JHDM2A) as a new miR-22-regulated gene. We show that KDM3A is overexpressed in Ewing Sarcoma, and that its depletion inhibits clonogenic and anchorage-independent growth in multiple patient-derived cell lines, and tumorigenesis in a xenograft model. KDM3A depletion further results in augmentation of the levels of the repressive H3K9me2 histone mark, and downregulation of pro-oncogenic factors in Ewing Sarcoma. Together, our studies identify the histone demethylase KDM3A as a new, miR-regulated, tumor promoter in Ewing Sarcoma. Show less

Dishevelled, a phosphoprotein scaffold, is a central component in all the Wnt-sensitive signaling pathways. In the present study, we report that Dishevelled is post-translationally modified, both in vitro and in vivo, via arginine methylation. We also show protein arginine methyl transferases 1 and 7 as the key enzymes catalyzing Dishevelled methylation. Interestingly, Wnt3a stimulation of F9 teratocarcinoma cells results in reduced Dishevelled methylation. Similarly, the methylation-deficient… Show more

Dishevelled, a phosphoprotein scaffold, is a central component in all the Wnt-sensitive signaling pathways. In the present study, we report that Dishevelled is post-translationally modified, both in vitro and in vivo, via arginine methylation. We also show protein arginine methyl transferases 1 and 7 as the key enzymes catalyzing Dishevelled methylation. Interestingly, Wnt3a stimulation of F9 teratocarcinoma cells results in reduced Dishevelled methylation. Similarly, the methylation-deficient mutant of Dishevelled, R271K, displayed spontaneous membrane localization and robust activation of Wnt signaling; suggesting that differential methylation of Dishevelled plays an important role in Wnt signaling. Thus arginine methylation is shown to be an important switch in regulation of Dishevelled function and Wnt signaling. Show less

Wnt7a is known to be a tumor suppressor that is lost in NSCLC, but no mechanism of loss has been established. Methylation of promoter regions has been established as a common mechanism of loss of tumor suppressor expression in NSCLC. We previously demonstrated that loss of Wnt7a in non-transformed lung epithelial cell lines led to increased cell growth, altered 3-D culture growth, and increased migration. The Wnt7a promoter has a higher percentage of methylation in NSCLC tumor tissue compared… Show more

Wnt7a is known to be a tumor suppressor that is lost in NSCLC, but no mechanism of loss has been established. Methylation of promoter regions has been established as a common mechanism of loss of tumor suppressor expression in NSCLC. We previously demonstrated that loss of Wnt7a in non-transformed lung epithelial cell lines led to increased cell growth, altered 3-D culture growth, and increased migration. The Wnt7a promoter has a higher percentage of methylation in NSCLC tumor tissue compared to matched normal lung tissue and methylation of the promoter region leads to decreased activity. We treated H157 and H1299 NSCLC cell lines with 5-Aza-2′-deoxycytidine and detected loss of Wnt7a promoter methylation, increased Wnt7a expression, and increased activity of the Wnt7a lung signaling pathway. When DNMT1 expression was knocked down by shRNA, expression of Wnt7a increased and methylation decreased. Together these data suggest that in NSCLC, Wnt7a is lost by methylation in a subset of tumors and that this methylation is maintained by DNMT1. Restoration of Wnt7a expression through demethylation could be an important therapeutic approach in the treatment of NSCLC. Show less

Sprouty proteins are potent receptor tyrosine kinase inhibitors that antagonize growth factor signaling and are involved in lung development. However, little is known about the regulation or targets of Sprouty-4 (Spry4) in lung cancer. Our study aimed to determine the role of Spry4 in NSCLC. We found that Spry4 mRNA expression was decreased in NSCLC cell lines and in dysplastic lung cell lines compared to a non-transformed cell line, suggesting that Spry4 has tumor suppressing activity. When… Show more

Sprouty proteins are potent receptor tyrosine kinase inhibitors that antagonize growth factor signaling and are involved in lung development. However, little is known about the regulation or targets of Sprouty-4 (Spry4) in lung cancer. Our study aimed to determine the role of Spry4 in NSCLC. We found that Spry4 mRNA expression was decreased in NSCLC cell lines and in dysplastic lung cell lines compared to a non-transformed cell line, suggesting that Spry4 has tumor suppressing activity. When Spry4 was stably transfected into H157 and H2122 NSCLC cell lines, decreased migration and invasion were observed. MMP-9 activity was decreased and expression of MMP inhibitors TIMP1 and CD82 were increased. Stable expression of Spry4 led to reduced cell growth and reduced anchorage independent growth in NSCLC cell lines, along with upregulation of tumor suppressors p53 and p21. Changes in epithelial and mesenchymal markers indicated that Spry4 expression induces a reversal of the epithelial to mesenchymal transition characteristic of tumor cells. Treatment of a non-transformed lung epithelial cell line with shRNA to Spry4 led to decreased expression of epithelial markers and increased cell growth, supporting the concept of Spry4 acting as a tumor suppressor. We demonstrated that activity of the Spry4 promoter is increased by Wnt7A/Fzd9 signaling through peroxisome proliferator activated receptor γ. These data present previously undescribed targets of Spry4 and suggest that Spry4 is a downstream target of Wnt7A/Fzd 9 signaling. Spry4 may have efficacy in the treatment of NSCLC. Show less

The goal of this study was to assess the ability of iloprost, an orally active prostacyclin analog, to inhibit transformed growth of human non-small cell lung cancer (NSCLC) and to define the mechanism of iloprost's tumor suppressive effects. In a panel of NSCLC cell lines, the ability of iloprost to inhibit transformed cell growth was not correlated with the expression of the cell surface receptor for prostacyclin, but instead was correlated with the presence of Frizzled 9 (Fzd 9) and the… Show more

The goal of this study was to assess the ability of iloprost, an orally active prostacyclin analog, to inhibit transformed growth of human non-small cell lung cancer (NSCLC) and to define the mechanism of iloprost's tumor suppressive effects. In a panel of NSCLC cell lines, the ability of iloprost to inhibit transformed cell growth was not correlated with the expression of the cell surface receptor for prostacyclin, but instead was correlated with the presence of Frizzled 9 (Fzd 9) and the activation of peroxisome proliferator-activated receptor-γ (PPARγ). Silencing of Fzd 9 blocked PPARγ activation by iloprost, and expression of Fzd 9 in cells lacking the protein resulted in iloprost's activation of PPARγ and inhibition of transformed growth. Interestingly, soluble Frizzled-related protein-1, a well-known inhibitor of Wnt/Fzd signaling, also blocked the effects of iloprost and Fzd 9. Moreover, mice treated with iloprost had reduced lung tumors and increased Fzd 9 expression. These studies define a novel paradigm, linking the eicosanoid pathway and Wnt signaling. In addition, these data also suggest that prostacyclin analogs may represent a new class of therapeutic agents in the treatment of NSCLC where the restoration of noncanonical Wnt signaling maybe important for the inhibition of transformed cell growth. Show less

The Wnt pathway is critical for normal development, and mutation of specific components is seen in carcinomas of diverse origins. The role of this pathway in lung tumorigenesis has not been clearly established. Recent studies from our laboratory indicate that combined expression of the combination of Wnt 7a and Frizzled 9 (Fzd 9) in Non-small Cell Lung Cancer (NSCLC) cell lines inhibits transformed growth. We have also shown that increased expression of peroxisome proliferator-activated… Show more

The Wnt pathway is critical for normal development, and mutation of specific components is seen in carcinomas of diverse origins. The role of this pathway in lung tumorigenesis has not been clearly established. Recent studies from our laboratory indicate that combined expression of the combination of Wnt 7a and Frizzled 9 (Fzd 9) in Non-small Cell Lung Cancer (NSCLC) cell lines inhibits transformed growth. We have also shown that increased expression of peroxisome proliferator-activated receptor gamma (PPARgamma) inhibits transformed growth of NSCLC and promotes epithelial differentiation of these cells. The goal of this study was to determine whether the effects of Wnt 7a/Fzd 9 were mediated through PPARgamma. We found that Wnt 7a and Fzd 9 expression led to increased PPARgamma activity. This effect was not mediated by altered expression of the protein. Wnt 7a and Fzd 9 expression resulted in activation of ERK5, which was required for PPARgamma activation in NSCLC. SR 202, a known PPARgamma inhibitor, blocked the increase in PPARgamma activity and restored anchorage-independent growth in NSCLC expressing Wnt 7a and Fzd 9. SR 202 also reversed the increase in E-cadherin expression mediated by Wnt 7a and Fzd 9. These data suggest that ERK5-dependent activation of PPARgamma represents a major effector pathway mediating the anti-tumorigenic effects of Wnt 7a and Fzd 9 in NSCLC. Show less

Lung cancer is a heterogeneous disease categorized into multiple subtypes of cancers which likely arise from distinct patterns of genetic alterations and disruptions. Precedent exists for a role of β-catenin, a downstream component of the Wnt signaling pathway that serves as a transcriptional co-activator with TCF/LEF, in several human cancers including colon carcinomas. In this study, we observed that β-catenin was highly and uniformly expressed in a panel of NSCLC cell lines and primary lung… Show more

Lung cancer is a heterogeneous disease categorized into multiple subtypes of cancers which likely arise from distinct patterns of genetic alterations and disruptions. Precedent exists for a role of β-catenin, a downstream component of the Wnt signaling pathway that serves as a transcriptional co-activator with TCF/LEF, in several human cancers including colon carcinomas. In this study, we observed that β-catenin was highly and uniformly expressed in a panel of NSCLC cell lines and primary lung tumors. By contrast, γ-catenin was weakly expressed or absent in several NSCLC cell lines and immunohistochemical analysis of primary NSCLC tumors revealed negligible to weak γ-catenin staining in ∼30% of the specimens. Treatment of NSCLC cells expressing reduced γ-catenin protein with 5-aza-2′-deoxycytidine (5aza2dc), a DNA methylation inhibitor, or trichostatin A (TSA), a histone deacetylase inhibitor, increased γ-catenin protein content in NSCLC cells with low γ-catenin expression. Significantly, the activity of a β-catenin/TCF-dependent luciferase reporter was markedly elevated in the NSCLC cell lines that underexpressed γ-catenin relative to those lines that highly expressed γ-catenin. Moreover, transfection of these cells with a γ-catenin expression plasmid reduced the elevated TCF activity by 85% and strongly inhibited cell growth on tissue culture plastic as well as anchorage-independent growth in soft agar. This study shows that γ-catenin can function as an inhibitor of β-catenin/TCF-dependent gene transcription and highlights γ-catenin as a potentially novel tumor suppressor protein in a subset of human NSCLC cancers. Show less