John Di Guglielmo

Abstract 352: The role of p62 (SQTSM1) in the transforming growth factor β signaling pathway

Transforming growth factor beta (TGFβ) is a cytokine that regulates many cellular processes, including cellular adhesion, proliferation and apoptosis. Its canonical downstream effectors include Smad2/3 proteins, which are phosphorylated and then translocate to the nucleus to alter gene transcriptional programs and promote processes such as epithelial-to-mesenchymal transition (EMT). Previous studies in our lab have shown that atypical Protein Kinase C (aPKC) isoforms associate with TGFβ receptors and modulate receptor trafficking and signal transduction. An aPKC-associated protein, p62 (SQSTM1) has been implicated in TGFβ-dependent EMT, however the mechanisms remain unclear.Here, we investigate the localization of p62 and its potential roles in modulating TGFβ signaling via knockdown and overexpression studies. Using antibody feeding and immunofluorescence microscopy, we support previous findings showing that p62 localizes to late endosomes. In addition, using a co-immunoprecipitation approach, we observed that p62 may associate with TGFβ type II receptor. To explore the functional role of p62 in TGFβ signaling, we conducted protein silencing using siRNA. We observed TGFβ-independent decreases in E-cadherin expression. However these changes were independent of the phosphorylation status or nuclear translocation of Smad2.P62 also plays an important role in autophagy by targeting proteins for degradation. Recently, prolonged TGFβ stimulation has been shown to induce cellular autophagy. Therefore, it is in our interest to characterize the relationship between TGFβ-dependent EMT and autophagy, and to determine whether p62 has a regulatory role between these two processes. Citation Format: Evelyn Ng, Adrian Gunaratne, John Di Guglielmo. The role of p62 (SQTSM1) in the transforming growth factor β signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 352. doi:10.1158/1538-7445.AM2017-352

Abstract 3335: Role of TGF-beta type III receptor in cell migration, invasion, and proliferation

This study aims to investigate the role of Transforming Growth Factor-beta type III receptor on tumor metastasis through cell migration and invasion. Metastasis is responsible for 90% of cancer related deaths. An important step in the metastatic process is the epithelial to mesenchymal transition (EMT) of cancer cells, which is stimulated by TGFβ. The binding of TGFβ to its type II receptor (TβRII) triggers the phosphorylation of ser/thr kinase TGFβ type I receptor (TβRI), and receptor-regulated Smad (R-Smad) which translocates to the nucleus and alters gene transcription. The activity of this pathway is affected by receptor localization on the cell membrane, while the presence of TβRI/II complex in clathrin-coated pits propagates TGFβ signaling. However, TβRI/II complex relocation to membrane lipid rafts reduces signaling. A TGFβ receptor that lacks kinase activity, type III (TβRIII), regulates TGFβ signaling through multiple roles. Previous investigations have determined TβRIII sequesters TGFβ ligand to reduce signaling, while also presenting TGFβ to TβRII to increase signaling. TβRIII has also been shown to bind type I and type II TGFβ receptors independently, and reverse their membrane partitioning from membrane lipid rafts to clathrin-coated pits. This TβRIII induced relocation of TβRI/II increases receptor half-life, altering TGFβ signaling. Many cancers demonstrate increased expression of TβRIII, suggesting that this protein is influential in the metastatic pathway. Interestingly, conflicting studies have shown that metastasis is stimulated when TβRIII is either overexpressed or knocked-down. Overexpression of exogenous TβRIII and knockdown of endogenous TβRIII using siRNA were used to explore alterations in cell migration and invasion potential. TGFβ signaling, receptor expression, membrane partitioning, and protein interaction analyses were performed using Western blotting, sucrose-density ultracentrifugation, and immunoprecipitation techniques. Immunofluorescent transwell assays measured relative cell migration and invasion through Matrigel. Transfection of TβRIII demonstrated both the punctate and membrane localization of TβRIII, while inhibiting transwell migration. The ability of transfected cells to migrate and invade through transwell assays clarified the impact of TβRIII expression on metastatic processes. Finally, both the overexpression and silencing of TβRIII resulted in significantly different cancer cell migration and invasion profiles when compared to untransfected cell lines. TβRIII expression and activity modify cellular migratory and invasive potential through the modulation of the TGFβ signaling pathway, altering epithelial to mesenchymal transitioning and therefore tumor metastasis. Citation Format: Anthony Ziccarelli, John Di Guglielmo. Role of TGF-beta type III receptor in cell migration, invasion, and proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3335. doi:10.1158/1538-7445.AM2017-3335

Abstract 4427: Role of Smurf2 in regulation of Smad anchor necessary for receptor activation (SARA) in TGFβ-receptor signaling

Non-small cell lung cancer (NSCLC) is a major form of lung cancer and is the leading cause of cancer mortality in the western world. Transforming growth factor β (TGFβ) deregulation leads to many human diseases: TGFβ is a tumor suppressor in normal lung epithelium; however, it switches roles and promotes lung cancer metastasis in cancer cells. TGFβ induces cell proliferation, migration and invasion in NSCLC cells. The canonical pathway of TGFβ is initiated through binding of TGFβ ligand to transmembrane Ser/Thr kinase receptors, which propagate their signaling via receptor regulated R-Smads; proteins that function as intracellular effectors in the TGFβ pathway. Once activated, R-Smads form a complex with common (Co)-Smads that translocates into the nucleus to regulate transcriptional responses. Access of R-Smads to the activated receptor complex is regulated by an adaptor protein called Smad anchor for receptor activation (SARA). SARA facilitates the activation of Smads and allow efficient Smad signalling. In addition to R-Smads and Co-Smads, inhibitory Smads (I-Smads) regulate TGFβ signalling. I-Smads block the signalling by competing against R-Smads for the association with TβR complex or by targeting receptors for ubiquitin-mediate degradation. I-Smads recruit the E3 ubiquitin ligases, Smad ubiquitination regulatory factors (Smurfs), to catalyze degradation of the receptor complex. The overall aim of the project is to characterize SARA in TGFβ receptor signalling and study the interaction of SARA with different proteins involved in the pathway, such as Smurf2 and Smad7. We first examined how SARA, Smurf2 and Smad7 influence each other by performing a multi-combination transfection study. We also examined the interaction between SARA and Smurf2 through co-immunoprecipitation. We observed that the level of SARA decrease in the presence of Smurf2 and Smad7. In the presence of Smurf2 ligase inactive mutant and Smad7, the level of SARA is somewhat recovered. However, SARA does not directly interact with Smurf2. These data together suggest that SARA and Smurf2 influence each other in a very close manner. It also suggest that it could be a transient interaction. Characterizing SARA in TGFβ receptor signalling will provide us with possible drug targets for NSCLC. Citation Format: Sanghyun Lee, John M. Di Guglielmo. Role of Smurf2 in regulation of Smad anchor necessary for receptor activation (SARA) in TGFβ-receptor signaling. [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 4427.

Abstract LB-345: Acetylenic tricyclic bis-(cyano enone) interacts with cysteine residues of actin and inhibits non-small cell lung cancer cell migration

During epithelial-to-mesenchymal transition (EMT), epithelial cells lose their cell-cell junctions, apical-basal polarity, and reorganize their actin cytoskeleton. These changes, among others, increase the motility of individual cells and promote a highly invasive phenotype. The actin cytoskeleton is also key in cell migration and invasion, as the polymerization of actin at the leading edge provides the necessary driving force for translocation and protrusions. Therefore, the actin cytoskeleton is a potential target for inhibiting tumour progression. In this study we characterized a novel tri-cyclic synthetic triterpenoid derivative, TBE-31, in its ability to associate with actin and inhibit cell migration. Using a pull-down approach, we demonstrate that TBE-31 binds directly to actin. Furthermore, TBE-31 inhibited branched and linear actin polymerization in vitro, and stress fibre formation in cultured cells. Actin cytoskeleton reorganization and stress fiber formation was also inhibited in TGFβ-dependent EMT of non-small cell lung cancer cells. We next used SwissDock software to investigate the potential molecular interaction between TBE-31 and actin. Swissdock predicted that TBE-31 would have a high affinity for binding to the same cleft as Cytochalasin D, a bona-fide actin polymerization inhibitor. As triterpenoids associate with cysteine residues in target proteins, we generated cysteine to alanine mutants of actin and verified TBE-31 binding using pull-down assays. We identified that cysteine 374 is important for TBE-31 interaction with actin. Finally, we observed that TBE-31 inhibited fibroblast and non-small cell lung tumour cell migration with an IC50 of 1.0 and 2.5 μM, respectively. Taken together, our results suggest that TBE-31 targets actin polymerization to alter cell morphology and inhibit cell migration. Citation Format: Eddie Chan, Akira Saito, Tadashi Honda, John Di Guglielmo. Acetylenic tricyclic bis-(cyano enone) interacts with cysteine residues of actin and inhibits non-small cell lung cancer cell migration. [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 LB-345.

Abstract 524: Assessing the interaction between acetylenic tricyclic bis-(cyano enone) and cysteine residues of actin to inhibit non-small cell lung cancer cell migration

During epithelial-to-mesenchymal transition (EMT), epithelial cells lose their cell-cell junctions, apical-basal polarity, and reorganize their actin cytoskeleton. These changes, among others, increase the motility of individual cells and promote a highly invasive phenotype. The actin cytoskeleton is also key in cell migration and invasion, as the polymerization of actin at the leading edge provides the necessary driving force for translocation and protrusions. Therefore, the actin cytoskeleton is a potential target for inhibiting tumour progression. In this study we characterized a novel tri-cyclic synthetic triterpenoid derivative, TBE-31, in its ability to associate with actin and inhibit cell migration. Using a pull-down approach, we demonstrate that TBE-31 binds directly to actin. Furthermore, TBE-31 inhibited branched and linear actin polymerization in vitro, and stress fibre formation in cultured cells. Actin cytoskeleton reorganization and stress fiber formation was also inhibited in TGFβ-dependent EMT of non-small cell lung cancer cells. We next used SwissDock software to investigate the potential molecular interaction between TBE-31 and actin. Swissdock predicted that TBE-31 would have a high affinity for binding to the same cleft as Cytochalasin D, a bona-fide actin polymerization inhibitor. As triterpenoids associate with cysteine residues in target proteins, we generated cysteine to alanine mutants of actin and verified TBE-31 binding using pull-down assays. Finally, we observed that TBE-31 inhibited fibroblast and non-small cell lung tumor cell migration with an IC 50 of 1.0 and 2.5 μM, respectively. Taken together, our results suggest that TBE-31 targets actin polymerization to alter cell morphology and inhibit cell migration. Citation Format: Eddie Chan, Akira Saito, Tadashi Honda, John Di Guglielmo. Assessing the interaction between acetylenic tricyclic bis-(cyano enone) and cysteine residues of actin to inhibit non-small cell lung cancer cell migration. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 524. doi:10.1158/1538-7445.AM2015-524

Abstract 5008: The acetylenic tricyclic bis(cyanoeneone), TBE-31 inhibits non-small cell lung cancer cell migration through direct binding with actin

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA During metastasis tumor cells undergo epithelial to mesenchymal transition (EMT), where cell-cell junctions dissolve and actin stress fibers are formed. This transition unmasks the migratory and invasive potential of the tumor cells. Here we show that the tricyclic compound TBE-31 binds to purified actin as well as actin from cell lysates. Furthermore, TBE-31 inhibits linear and branched actin polymerization in vitro as well as stress fiber formation in fibroblasts. We also observed that TBE-31 inhibits stress fiber formation in non-small lung cancer cells during TGFβ-dependent EMT. Interestingly, TBE-31 does not interfere with TGFβ-dependent signaling or changes in E- and N-cadherin protein levels during EMT. Finally, we observed that TBE-31 inhibits non-small cell lung tumor cell migration. Our results suggest that TBE-31 targets linear actin polymerization to alter cell morphology and inhibit cell migration. Citation Format: Eddie Chan, Akira Saito, Tadashi Honda, John Di Guglielmo. The acetylenic tricyclic bis(cyanoeneone), TBE-31 inhibits non-small cell lung cancer cell migration through direct binding with actin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5008. doi:10.1158/1538-7445.AM2014-5008

Abstract 476: The effects of the synthetic triterpenoid acetylenic tricyclic bis-(cyano enone) on cell migration and epithelial-to-mesenchymal transition

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL During metastasis, epithelial tumor cells undergo epithelial to mesenchymal transition (EMT) allowing them to migrate to distant organs to establish secondary tumors. Cell migration is initiated by the asymmetrical localization of polarity proteins towards the leading edge. This stimulates the reorganization and polymerization of the microtubule and actin cytoskeletons, which form protrusions in the plasma membrane to drive the cell forwards. Metastasis accounts for over 90% of cancer related deaths, thus a reduction in cancer mortality will require therapies aimed at preventing or delaying the events of metastasis. Our lab has identified the synthetic triterpenoid, CDDO-Im, as a potent inhibitor of cell migration by inhibiting the polymerization of branched actin and disrupting the organization of the microtubule network (To et al., J Biol Chem. 2008, 2010). Recently, TBE-31 has been developed, which is a smaller 3-ring compound that contains the same active functional groups as CDDO-Im. Here we assessed the effects of TBE-31 on cell migration and EMT. TBE-31 was found to displace the polarity proteins from the leading edge of migrating cells and induce multiple cell protrusions. The microtubule network was also observed to be disorganized and the rate of polymerization for both tubulin and actin were lowered in the presence of TBE-31. These effects are believed to allow submicromolar concentrations TBE-31 to inhibit cell migration by as much as 60%. Finally, the reorganization of actin and formation of stress fibers normally associated with EMT was also inhibited by TBE-31 in A549 tumor cells. We are now confirming our results in a panel of lung tumor cell lines. In conclusion, TBE-31 disrupts the cytoskeleton and inhibits cell migration and preliminary data suggest TBE-31 may also hinder EMT. Further research will be needed to determine the overall impact these effects have on the progression of metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 476. doi:1538-7445.AM2012-476

Abstract 1400: Inhibition of cell migration by the synthetic triterpenoid acetylenic tricyclic bis-(cyano enone)

Triterpenoids are a family of naturally occurring compounds that have multiple anti-tumorigenic effects, including the ability to induce cell differentiation, apoptosis and inhibit proliferation. Research in our lab has demonstrated that a synthetically derived 5 ring triterpenoid, 2-cyano-3, 12-dioxooleana-1,9-dien-28-oic acid-imidazolide (CDDO-Im), inhibits cell migration (To et al., 2008, J. Biol. Chem. 283:11700), which is essential in numerous physiological processes including tumor metastasis. Recently, a smaller 3 ring compound containing the same active functional groups as CDDO-Im has been derived; acetylenic tricyclic bis-(cyano enone) [(+/-)-(4aa,8aa,10ab)-1,2,4a,6,8a,9,10,10a-octahydro-8a-ethynyl-1,1,4a-trimethyl-2,6 dioxophenanthrene-3,7-dicarbonitrile] (TBE-31). Here we examined the effect of TBE-31 on cell migration. Using scratch assays with Rat2 fibroblasts we demonstrated that concentrations of TBE-31 above 0.75 µM were effective in inhibiting fibroblast migration. To elucidate the mechanism(s) for this inhibition we examined different components of the cytoskeleton and the polarity complex, which are both crucial for cell migration. To examine cell polarity, we used immunofluorescence microscopy to assess the localization of two proteins involved in the polarization of migrating cells, IQGAP1 and RAC1. Similar to CDDO-Im, TBE-31 was observed to reduce the elongation and polarity of cells, however unlike CDDO-Im, IQGAP1 and RAC1 in TBE-31 treated cells were localized in punctate fashion around the cell perimeter instead of at the leading edge. Using immunofluorescence imaging, the cytoskeleton of Mv1Lu mink lung cells or COS7 cells treated with TBE-31 were analyzed. The microtubule network of treated cells displayed a unique cross-hatched phenotype and were resistant to nocodazole depolymerization. In addition, the intermediate filament cytoskeleton was observed to retract towards the nucleus. Lastly, in vitro actin polymerization assays revealed that TBE-31 inhibits linear but not branched actin polymerization. We conclude that TBE-31 is an effective inhibitor of cell migration and further research will shed light on the mechanisms that TBE-31 uses to modulate the cell migration machinery. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1400. doi:10.1158/1538-7445.AM2011-1400

Abstract 1967: Ligand-dependent TGFβ signalling potential

Transforming growth factor beta (TGFβ) is a cytokine which regulates many normal biological processes such as organogenesis and cell cycle control. Its signaling pathway is under intense study as it is commonly dysregulated in pathophysiological conditions such as cancer and fibrosis. The TGFβ signalling cascade is initiated by the binding of ligand to cell-surface serine-threonine kinase receptors. Binding of ligand to the TGFβ receptors initiates phosphorylation of intracellular signalling proteins called Smads, which can then enter the nucleus and initiate specific transcriptional programs. There are three TGFβ ligands which can activate canonical Smad signalling: TGFβ1, TGFβ2 and TGFβ3. These ligands share significant sequence homology (70-80%) but have different spatial and temporal patterns of expression in development. Although TGFβ1 and TGFβ3 are highly expressed in the tumour microenvironment, the role of TGFβ3 has been largely over-looked and thought to be the same as TGFβ1. The objective of the present study is to evaluate the signalling capacity of TGFβ1 and TGFβ3 in non-small cell lung cancer cells. Using dose response studies, we show that TGFβ1 ligand induces Smad2 phosphorylation to a greater extent than TGFβ3. We also show that TGFβ3 induces a lower reduction in steady-state levels of E-cadherin, a protein involved in cell-cell adhesion, compared to TGFβ1. To evaluate the transcriptional programs of both ligands, we used microarray technology to assess gene transcription in non-small cell lung cancer cells treated with TGFβ1 or TGFβ3. Consistent with our signalling data, we found that TGFβ3 induces far fewer genes than TGFβ1. Future studies will evaluate the differential roles of TGFβ1 and TGFβ3 in cancer cell migration and epithelial-to-mesenchymal transition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1967. doi:10.1158/1538-7445.AM2011-1967

Abstract 1051: Atypical PKC and TGFβ receptors co-operate to phosphorylate Par6

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Transforming growth factor beta (TGFβ) is involved in many aspects of cellular behavior including apoptosis and differentiation. In tumor cells, TGFβ signaling alters cell plasticity through a loss in apical-basal cell polarity and induces epithelial to mesenchymal transition (EMT). Integral to this process is the phosphorylation of a polarity protein, Par6, on a conserved serine residue (S345). Recent work has shown that phosphorylation of this site not only increases cell plasticity and EMT, but also promotes an invasive phenotype in breast cancer cells. Our work suggests that a polarity partner of Par6, atypical Protein Kinase C (aPKC), can also phosphorylate Par6 on this critical site to drive distinct cellular changes in non-small cell lung cancer (NSCLC) cells. Using co-immunoprecipitation studies and immunofluorescence microscopy, we show that aPKCι interacts with TGFβ receptors through the adaptor Par6, and that these proteins localize to the leading edge of migrating cells. Furthermore, co-expression of Par6 with TGFβ receptors results in increased phospho-Par6 levels as previously reported. However, we have also found that phosphorylation of Par6 increases in the presence of aPKC. aPKC kinase activity as well as association with Par6 were found to be important for Par6 phosphorylation as evidenced by a loss in phospho-Par6 using a kinase deficient mutant of PKCζ (PKCζ -KR) as well as a mutant of Par6 that does not bind aPKC (Par6-K19A). Furthermore, gene silencing of atypical PKC reduces TGFβ induced cell morphology changes, E-cadherin loss, and stress fiber production. Finally, gene silencing of atypical PKC was observed to reduce TGFβ dependent migration of NSCLC cells. In conclusion, our results indicate that atypical PKC cooperate with TGFβ receptors to regulate phospho-Par6 levels to drive an invasive phenotype of NSCLC cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1051. doi:10.1158/1538-7445.AM2011-1051