Somesh Baranwal

Nonredundant roles of cytoplasmic β- and γ-actin isoforms in regulation of epithelial apical junctions.

The functional effects of cytoplasmic actins on epithelial junctions are examined by using isoform-specific siRNAs and cell-permeable inhibitory peptides. Unique roles of cytoplasmic actin isoforms in regulating structure and remodeling of adherens and tight junctions are revealed.

Novel mechanism of cytokine-induced disruption of epithelial barriers: Janus kinase and protein kinase D-dependent downregulation of junction protein expression.

The ductal epithelium plays a key role in physiological secretion of pancreatic enzymes into the digestive system. Loss of barrier properties of the pancreatic duct may contribute to the development of pancreatitis and metastatic dissemination of pancreatic tumors. Proinflammatory cytokines are essential mediators of pancreatic inflammation and tumor progression; however, their effects on the integrity and barrier properties of the ductal epithelium have not been previously addressed. In the present study, we investigate mechanisms of cytokine-induced disassembly of tight junctions (TJs) and adherens junctions (AJs) in a model pancreatic epithelium. Exposure of HPAF-II human pancreatic epithelial cell monolayers to interferon (IFN)γ disrupted integrity and function of apical junctions as manifested by increased epithelial permeability and cytosolic translocation of AJ and TJ proteins. Tumor necrosis factor (TNF)α potentiated the effects of IFNγ on pancreatic epithelial junctions. The cytokine-induced increase in epithelial permeability and AJ/TJ disassembly was attenuated by pharmacological inhibition of Janus kinase (JAK) and protein kinase D (PKD). Loss of apical junctions in IFNγ/TNFα-treated HPAF-II cells was accompanied by JAK and PKD dependent decrease in expression of AJ (E-cadherin, p120 catenin) and TJ (occludin, ZO-1) proteins. Depletion of E-cadherin or p120 catenin recapitulated the effects of cytokines on HPAF-II cell permeability and junctions. Our data suggests that proinflammatory cytokines disrupt pancreatic epithelial barrier via expressional downregulation of key structural components of AJs and TJs. This mechanism is likely to be important for pancreatic inflammatory injury and tumorigenesis.

Synthetic, non-saccharide, glycosaminoglycan mimetics selectively target colon cancer stem cells.

Selective targeting of cancer stem-like cells (CSCs) is a paradigm-shifting approach. We hypothesized that CSCs can be targeted by interfering with functions of sulfated glycosaminoglycans, which play key roles in cancer cell growth, invasion and metastasis. We developed a tandem, dual screen strategy involving (1) assessing inhibition of monolayer versus spheroid growth and (2) assessing inhibition of primary versus secondary spheroid growth to identify G2.2, a unique sulfated nonsaccharide GAG mimetic (NSGM) from a focused library of 53 molecules, as a selective inhibitor of colon CSCs. The NSGM down-regulated several CSC markers through regulation of gene transcription, while closely related, inactive NSGMs G1.4 and G4.1 demonstrated no such changes. G2.2’s effects on CSCs were mediated, in part, through induction of apoptosis and inhibition of self-renewal factors. Overall, this work presents the proof-of-principle that CSCs can be selectively targeted through novel NSGMs, which are likely to advance fundamental understanding on CSCs while also aiding development of novel therapeutic agents.

Loss of γ-cytoplasmic actin triggers myofibroblast transition of human epithelial cells

Loss of γ-cytoplasmic actin induces epithelial-to-myofibroblast transition (EmyT), which depends on activation of SRF and its cofactor, MRTF, formin-mediated actin polymerization, and activated Rho GTPase. This demonstrates a unique role of γ-cytoplasmic actin in regulating the epithelial phenotype and the suppression of EmyT.

Actin-interacting protein 1 controls assembly and permeability of intestinal epithelial apical junctions.

Adherens junctions (AJs) and tight junctions (TJs) are crucial regulators of the integrity and restitution of the intestinal epithelial barrier. The structure and function of epithelial junctions depend on their association with the cortical actin cytoskeleton that, in polarized epithelial cells, is represented by a prominent perijunctional actomyosin belt. The assembly and stability of the perijunctional cytoskeleton is controlled by constant turnover (disassembly and reassembly) of actin filaments. Actin-interacting protein (Aip) 1 is an emerging regulator of the actin cytoskeleton, playing a critical role in filament disassembly. In this study, we examined the roles of Aip1 in regulating the structure and remodeling of AJs and TJs in human intestinal epithelium. Aip1 was enriched at apical junctions in polarized human intestinal epithelial cells and normal mouse colonic mucosa. Knockdown of Aip1 by RNA interference increased the paracellular permeability of epithelial cell monolayers, decreased recruitment of AJ/TJ proteins to steady-state intercellular contacts, and attenuated junctional reassembly in a calcium-switch model. The observed defects of AJ/TJ structure and functions were accompanied by abnormal organization and dynamics of the perijunctional F-actin cytoskeleton. Moreover, loss of Aip1 impaired the apico-basal polarity of intestinal epithelial cell monolayers and inhibited formation of polarized epithelial cysts in 3-D Matrigel. Our findings demonstrate a previously unanticipated role of Aip1 in regulating the structure and remodeling of intestinal epithelial junctions and early steps of epithelial morphogenesis.

Actin-Depolymerizing Factor and Cofilin-1 Have Unique and Overlapping Functions in Regulating Intestinal Epithelial Junctions and Mucosal Inflammation

The actin cytoskeleton is a crucial regulator of the intestinal mucosal barrier, controlling the assembly and function of epithelial adherens and tight junctions (AJs and TJs). Junction-associated actin filaments are dynamic structures that undergo constant turnover. Members of the actin-depolymerizing factor (ADF) and cofilin protein family play key roles in actin dynamics by mediating filament severing and polymerization. We examined the roles of ADF and cofilin-1 in regulating the structure and functions of AJs and TJs in the intestinal epithelium. Knockdown of either ADF or cofilin-1 by RNA interference increased the paracellular permeability of human colonic epithelial cell monolayers to small ions. Additionally, cofilin-1, but not ADF, depletion increased epithelial permeability to large molecules. Loss of either ADF or cofilin-1 did not affect the steady-state morphology of AJs and TJs but attenuated de novo junctional assembly. The observed defects in AJ and TJ formation were accompanied by delayed assembly of the perijunctional filamentous actin belt. A total loss of ADF expression in mice did not result in a defective mucosal barrier or in spontaneous gut inflammation. However, ADF-null mice demonstrated increased intestinal permeability and exaggerated inflammation during dextran sodium sulfate-induced colitis. Our findings demonstrate novel roles for ADF and cofilin-1 in regulating the remodeling and permeability of epithelial junctions, as well as the role of ADF in limiting the severity of intestinal inflammation.

Inhibition of C-terminal binding protein attenuates transcription factor 4 signaling to selectively target colon cancer stem cells

Selective targeting of cancer stem cells (CSCs), implicated in tumor relapse, holds great promise in the treatment of colorectal cancer. Overexpression of C-terminal binding protein (CtBP), an NADH dependent transcriptional regulator, is often observed in colon cancer. Of note, TCF-4 signaling is also up-regulated in colonic CSCs. We hypothesized that CtBP, whose dehydrogenase activity is amenable to pharmacological inhibition by 4-methylthio-2-oxobutyric acid (MTOB), positively regulates TCF-4 signaling, leading to CSC growth and self-renewal. CSCs demonstrated significant upregulation of CtBP1 and CtBP2 levels (mRNA and protein) and activity partly due to increased NADH/NAD ratio, as well as increased TCF/LEF transcriptional activity, compared to respective controls. Depletion of CtBP2 inhibited, while its overexpression enhanced, CSC growth (1° spheroids) and self-renewal (2°/3° spheroids). Similarly, MTOB caused a robust inhibition of spheroid growth and self-renewal in a dose dependent manner. MTOB displayed significantly greater selectivity for growth inhibition in the spheroids, at least in part through induction of apoptosis, compared to monolayer controls. Moreover, MTOB inhibited basal as well as induced (by GSK-3β inhibitor) TCF/LEF activity while suppressing mRNA and protein levels of several β-catenin target genes (CD44, Snail, C-MYC and LGR5). Lastly, CtBP physically interacted with TCF-4, and this interaction was significantly inhibited in the presence of MTOB. The above findings point to a novel role of CtBPs in the promotion of CSC growth and self-renewal through direct regulation of TCF/LEF transcription. Moreover, small molecular inhibition of its function can selectively target CSCs, presenting a novel approach for treatment of colorectal cancer focused on targeting of CSCs.

Heparan sulfate hexasaccharide selectively inhibits cancer stem cells self-renewal by activating p38 MAP kinase

Heparan sulfate (HS) plays a role in the majority of essential hallmarks of cancer, yet its ability to modulate self-renewal, especially of cancer stem cells (CSCs), remains unknown. We have discovered that a non-anticoagulant HS hexasaccharide (HS06) sequence, but not other shorter or longer sequences, selectively inhibited CSC self-renewal and induced apoptosis in colorectal, pancreatic, and breast CSCs suggesting a very general phenomenon. HS06 inhibition of CSCs relied upon early and sustained activation of p38α/β mitogen activated protein kinase (MAPK) but not other MAPKs family members i.e. ERK and JNK. In contrast, polymeric HS induced exactly opposite changes in MAPK activation and failed to inhibit CSCs. In fact, TCF4 signaling, a critical regulator of CSC self-renewal, was inhibited by HS06 in a p38 activation dependent fashion. In conclusion, HS06 selectively inhibits CSCs self-renewal by causing isoform specific activation of p38MAPK to inhibit TCF4 signaling. These observations on chain length-induced specificity carry major mechanistic implications with regard to HS in cancer biology, while also presenting a novel paradigm for developing novel anti-CSC hexasaccharides that prevent cancer relapse.

A Strategic Approach to Identification of Selective Inhibitors of Cancer Stem Cells

Cancer stem-like cells (CSC) have been implicated in resistance to conventional chemotherapy as well as invasion and metastasis resulting in tumor relapse in majority of epithelial cancers including colorectal cancer. Hence, targeting CSC by small molecules is likely to improve therapeutic outcomes. Glycosaminoglycans (GAGs) are long linear polysaccharide molecules with varying degrees of sulfation that allows specific GAG-protein interaction which plays a key role in regulating cancer hallmarks such as cellular growth, angiogenesis, and immune modulation. However, identifying selective CSC-targeting GAG mimetic has been marred by difficulties associated with isolating and enriching CSC in vitro. Herein, we discuss two distinct methods, spheroid growth and EMT-transformed cells, to enrich CSC and set up medium- and high-throughput screen to identify selective CSC-targeting agents.

395 Synthetic, Non-Saccharide Glycosaminoglycan Mimetics Selectively Target Colon Cancer Stem Cells

Selective targeting of cancer stem-like cells (CSCs) is a paradigm-shifting approach. We hypothesized that CSCs can be targeted by interfering with functions of sulfated glycosaminoglycans, which play key roles in cancer cell growth, invasion and metastasis. We developed a tandem, dual screen strategy involving (1) assessing inhibition of monolayer versus spheroid growth and (2) assessing inhibition of primary versus secondary spheroid growth to identify G2.2, a unique sulfated nonsaccharide GAG mimetic (NSGM) from a focused library of 53 molecules, as a selective inhibitor of colon CSCs. The NSGM down-regulated several CSC markers through regulation of gene transcription, while closely related, inactive NSGMs G1.4 and G4.1 demonstrated no such changes. G2.2’s effects on CSCs were mediated, in part, through induction of apoptosis and inhibition of self-renewal factors. Overall, this work presents the proof-of-principle that CSCs can be selectively targeted through novel NSGMs, which are likely to advance ...