Basabi Rana

Cell-extracellular matrix interactions can regulate the switch between growth and differentiation in rat hepatocytes: reciprocal expression of C/EBP alpha and immediate-early growth response transcription factors.

Previous investigations have shown that culture of freshly isolated hepatocytes under conventional conditions, i.e., on dried rat tail collagen in the presence of growth factors, facilitates cell growth but also causes an extensive down-regulation of most liver-specific functions. This dedifferentiation process can be prevented if the cells are cultured on a reconstituted basement membrane gel matrix derived from the Englebreth-Holm-Swarm mouse sarcoma tumor (EHS gel). To gain insight into the mechanisms regulating this response to extracellular matrix, we are analyzing the activities of two families of transcription factors, C/EBP and AP-1, which control the transcription of hepatic and growth-responsive genes, respectively. We demonstrate that isolation of hepatocytes from the normal quiescent rat liver by collagenase perfusion activates the immediate-early growth response program, as indicated by increased expression of c-jun, junB, c-fos, and c-myc mRNAs. Adhesion of these activated cells to dried rat tail collagen augments the elevated levels of these mRNAs for the initial 1 to 2 h postplating; junB and c-myc mRNA levels then drop steeply, with junB returning to normal quiescence and the c-myc level remaining slightly elevated during the 3-day culture period. Levels of c-jun mRNA and AP-1 DNA binding activity, however, remain elevated from the outset, while C/EBP alpha mRNA expression is down-regulated, resulting in a decrease in the steady-state levels of the 42- and 30-kDa C/EBP alpha polypeptides and C/EBP alpha DNA binding activity. In contrast, C/EBP beta mRNA production remains at near-normal hepatic levels for 5 to 8 days of culture, although its DNA binding activity decreases severalfold during this time. Adhesion of hepatocytes to the EHS gel for the same period of time dramatically alters this program: it arrests growth and inhibits AP-1 DNA binding activity and the expression of c-jun, junB, and c-myc mRNAs, but, in addition, it restores C/EBP alpha mRNA and protein as well as C/EBP alpha and C/EBP beta DNA binding activities to the abundant levels present in freshly isolated hepatocytes. These changes are not due merely to growth inhibition, because suppression of hepatocyte proliferation on collagen by epidermal growth factor starvation or addition of transforming growth factor beta does not inhibit AP-1 activity or restore C/EBP alpha DNA binding activity to normal hepatic levels. These data suggest that expression of the normal hepatic phenotype requires that hepatocytes exist in a G0 state of growth arrest, facilitated here by adhesion of cells to the EHS gel, in order to express high levels of hepatic transcription factors such as C/EBP alpha.

Growth-dependent inhibition of CCAAT enhancer-binding protein (C/EBP alpha) gene expression during hepatocyte proliferation in the regenerating liver and in culture.

As an approach to understanding physiological mechanisms that control the proliferation of highly differentiated cells, we are addressing whether certain hepatic transcription factors participate in mechanisms that control the growth of hepatocytes. We have focused on CCAAT enhancer-binding protein (C/EBP alpha), a transcription factor which is highly abundant in normal liver and is considered to regulate expression of many genes, including some involved in energy metabolism (S. L. McKnight, M. D. Lane, and S. Gluecksohn-Walsh. Genes Dev. 3:2021-2024, 1989). Using Northern (RNA) blot analysis, we have examined the expression of C/EBP alpha mRNA during liver regeneration and in primary cultures of hepatocytes. C/EBP alpha mRNA levels decrease 60 to 80% within 1 to 3 h after partial hepatectomy as the cells move from G0 to G1 and decrease further when cells progress into S phase. Run-on transcription analysis is in agreement with the Northern blot data, thus suggesting that C/EBP alpha is transcriptionally regulated in regenerating liver. C/EBP alpha mRNA expression also decreases dramatically during the growth of freshly isolated normal hepatocytes cultured under conventional conditions (on dried rat tail collagen; stimulated to proliferate by epidermal growth factor [EGF] and insulin). Cultures of hepatocytes on rat tail collagen in the presence or absence of EGF clearly show that within 3 h, EGF depresses C/EBP alpha mRNA expression and that this effect is substantially greater by 4 h. Inhibition of protein synthesis in the liver by cycloheximide or in cultured hepatocytes by puromycin or cycloheximide effectively blocks the down-regulation of C/EBP alpha gene expression, apparently by stabilizing the normal rapid turnover of the C/EBP alpha mRNA (half-life of <2 h). This drop in C/EBP alpha gene expression in response to activation of hepatocyte growth is consistent with the proposal that C/EBP alpha has an antiproliferative role to play in highly differentiated cells (R. M. Umek, A. D. Friedman, and S. L. McKnight, Science 251: 288-292, 1991).

Glycogen Synthase Kinase-3β Induces Neuronal Cell Death via Direct Phosphorylation of Mixed Lineage Kinase 3

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase member that activates the c-Jun N-terminal kinase (JNK) pathway. Aberrant activation of MLK3 has been implicated in neurodegenerative diseases. Similarly, glycogen synthase kinase (GSK)-3β has also been shown to activate JNK and contribute to neuronal apoptosis. Here, we show a functional interaction between MLK3 and GSK-3β during nerve growth factor (NGF) withdrawal-induced cell death in PC-12 cells. The protein kinase activities of GSK-3β, MLK3, and JNK were increased upon NGF withdrawal, which paralleled increased cell death in NGF-deprived PC-12 cells. NGF withdrawal-induced cell death and MLK3 activation were blocked by a GSK-3β-selective inhibitor, kenpaullone. However, the MLK family inhibitor, CEP-11004, although preventing PC-12 cell death, failed to inhibit GSK-3β activation, indicating that induction of GSK-3β lies upstream of MLK3. In GSK-3β-deficient murine embryonic fibroblasts, ultraviolet light was unable to activate MLK3 kinase activity, a defect that was restored upon ectopic expression of GSK-3β. The activation of MLK3 by GSK-3β occurred via phosphorylation of MLK3 on two amino acid residues, Ser789 and Ser793, that are located within the C-terminal regulatory domain of MLK3. Furthermore, the cell death induced by GSK-3β was mediated by MLK3 in a manner dependent on its phosphorylation of the specific residues within the C-terminal domain by GSK-3β. Taken together, our data provide a direct link between GSK-3β and MLK3 activation in a neuronal cell death pathway and identify MLK3 as a direct downstream target of GSK-3β. Inhibition of GSK-3 is thus a potential therapeutic strategy for neurodegenerative diseases caused by trophic factor deprivation.

Caspase-mediated Cleavage of β-Catenin Precedes Drug-induced Apoptosis in Resistant Cancer Cells

A delicate balance between cell death and survival pathways maintains normal physiology, which is altered in many cancers, shifting the balance toward increased survival. Several studies have established a close connection between the Wnt/β-catenin pathway and tumorigenesis, aberrant activation of which might contribute toward increased cancer cell growth and survival. Extensive research is underway to identify therapeutic agents that can induce apoptosis specifically in cancer cells with minimal collateral damage to normal cells. Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis specifically in tumor cells, many cancer cells develop resistance, which can be overcome by combinatorial treatment with other agents: for example, peroxisome proliferator-activated receptorγ (PPARγ) ligands. To identify the molecular target mediating combinatorial drug-induced apoptosis, we focused on β-catenin, a protein implicated in oncogenesis. Our results show that co-treatment of TRAIL-resistant cancer cells with TRAIL and the PPARγ ligand troglitazone leads to a reduction ofβ-catenin expression, coinciding with maximal apoptosis. Modulation of β-catenin levels via ectopic overexpression or small interference RNA-mediated gene silencing modulates drug-induced apoptosis, indicating involvement of β-catenin in regulating this pathway. More in-depth studies indicated a post-translational mechanism, independent of glycogen synthase kinase-3β activity regulating β-catenin expression following combinatorial drug treatment. Furthermore, TRAIL- and troglitazone-induced apoptosis was preceded by a cleavage of β-catenin, which was complete in a fully apoptotic population, and was mediated by caspases-3 and -8. These results demonstrate β-catenin as a promising new target of drug-induced apoptosis, which can be targeted to sensitize apoptosis-resistant cancer cells.

TRAF2-MLK3 interaction is essential for TNF-α-induced MLK3 activation

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase that is activated by tumor necrosis factor-α (TNF-α) and specifically activates c-Jun N-terminal kinase (JNK) on TNF-α stimulation. The mechanism by which TNF-α activates MLK3 is still not known. TNF receptor-associated factors (TRAFs) are adapter molecules that are recruited to cytoplasmic end of TNF receptor and mediate the downstream signaling, including activation of JNK. Here, we report that MLK3 associates with TRAF2, TRAF5 and TRAF6; however only TRAF2 can significantly induce the kinase activity of MLK3. The interaction domain of TRAF2 maps to the TRAF domain and for MLK3 to its C-terminal half (amino acids 511-847). Endogenous TRAF2 and MLK3 associate with each other in response to TNF-α treatment in a time-dependent manner. The association between MLK3 and TRAF2 mediates MLK3 activation and competition with the TRAF2 deletion mutant that binds to MLK3 attenuates MLK3 kinase activity in a dose-dependent manner, on TNF-α treatment. Furthermore the downstream target of MLK3, JNK was activated by TNF-α in a TRAF2-dependent manner. Hence, our data show that the direct interaction between TRAF2 and MLK3 is required for TNF-α-induced activation of MLK3 and its downstream target, JNK.

Mixed Lineage Kinase 3 Modulates β-Catenin Signaling in Cancer Cells

Background: β-Catenin mediates a wide variety of cellular processes, but the signaling pathways regulating β-catenin downstream events are not fully understood. The role of MLK3 in modulating β-catenin pathway has not been reported earlier. Results: MLK3 can induce β-catenin stabilization but inhibit conventional β-catenin/TCF transcriptional activation. Conclusion: These provide a new mechanism of regulating β-catenin/TCF axis. Significance: MLK3 can be targeted in regulating the growth of β-catenin overexpressing tumors. Expression of β-catenin is strictly regulated in normal cells via the glycogen synthase kinase 3β (GSK3β)- adenomatous polyposis coli-axin-mediated degradation pathway. Mechanisms leading to inactivation of this pathway (example: activation of Wnt/β-catenin signaling or mutations of members of the degradation complex) can result in β-catenin stabilization and activation of β-catenin/T-cell factor (TCF) signaling. β-Catenin-mediated cellular events are diverse and complex. A better understanding of the cellular signaling networks that control β-catenin pathway is important for designing effective therapeutic strategies targeting this axis. To gain more insight, we focused on determining any possible cross-talk between β-catenin and mixed lineage kinase 3 (MLK3), a MAPK kinase kinase member. Our studies indicated that MLK3 can induce β-catenin expression via post-translational stabilization in various cancer cells, including prostate cancer. This function of MLK3 was dependent on its kinase activity. MLK3 can interact with β-catenin and phosphorylate it in vitro. Overexpression of GSK3β-WT or the S9A mutant was unable to antagonize MLK3-induced stabilization, suggesting this to be independent of GSK3β pathway. Surprisingly, despite stabilizing β-catenin, MLK3 inhibited TCF transcriptional activity in the presence of both WT and S37A β-catenin. These resulted in reduced expression of β-catenin/TCF downstream targets Survivin and myc. Immunoprecipitation studies indicated that MLK3 did not decrease β-catenin/TCF interaction but promoted interaction between β-catenin and KLF4, a known repressor of β-catenin/TCF transcriptional activity. In addition, co-expression of MLK3 and β-catenin resulted in significant G2/M arrest. These studies provide a novel insight toward the regulation of β-catenin pathway, which can be targeted to control cancer cell proliferation, particularly those with aberrant activation of β-catenin signaling.

Transcriptional regulation of mixed lineage kinase 3 by estrogen and its implication in ER-positive breast cancer pathogenesis

Mixed Lineage Kinase 3 (MLK3), also called as MAP3K11 is a tightly regulated MAP3K member but its cellular function is still not fully understood. Earlier we reported post-translational regulation of MLK3 by estrogen (E2) that inhibited the kinase activity and favored survival of ER+ breast cancer cells. Here we report that MLK3 is also transcriptionally downregulated by E2 in ER+ breast cancer cells. Publicly available data and in situ hybridization of human breast tumors showed significant down regulation of MLK3 transcripts in ER+ tumors. The basal level of MLK3 transcripts and protein in ER+ breast cancer cell lines were significantly lower, and the protein expression was further down regulated by E2 in a time-dependent manner. Analysis of the promoter of MLK3 revealed two ERE sites which were regulated by E2 in ER+ but not in ER− breast cancer cell lines. Both ERα and ERβ were able to bind to MLK3 promoter and recruit nuclear receptor co-repressors (NCoR, SMRT and LCoR), leading to down-regulation of MLK3 transcripts. Collectively these results suggest that recruitment of nuclear receptor co-repressor is a key feature of ligand-dependent transcriptional repression of MLK3 by ERs. Therefore coordinated transcriptional and post-translational repression of pro-apoptotic MLK3 probably is one of the mechanisms by which ER+ breast cancer cells proliferate and survive.

Modulation of glycogen synthase kinase-3β following TRAIL combinatorial treatment in cancer cells

Glycogen Synthase Kinase-3β (GSK3β) is a serine/threonine kinase, known to regulate various cellular processes including proliferation, differentiation, survival, apoptosis as well as TRAIL-resistance. Thus pathways that can modulate GSK3β axis are important targets for cancer drug development. Our earlier studies have shown that combinatorial treatment with Troglitazone (TZD) and TRAIL can induce apoptosis in TRAIL-resistant cancer cells. The current studies were undertaken to investigate whether GSK3β pathway was modulated during this apoptosis. Our results indicated an increase in inhibitory GSK3βSer9 phosphorylation during apoptosis, mediated via AKT. At a later time, however, TZD alone and TRAIL-TZD combination produced a dramatic reduction of GSK3β expression, which was abolished by cycloheximide. Luciferase assays with GSK3β-luc promoter reporter showed that TZD can effectively antagonize GSK3β promoter activity. Since TZD is a ligand for transcription factor PPARγ and can activate AMPK, we determined their roles on antagonism of GSK3β. Knockdown of PPARγ was unable to restore GSK3β expression or antagonize GSK3βSer9 phosphorylation. Although pretreatment with Compound C (pharmacological inhibitor of AMPK) partially rescued GSK3β expression, knockdown of AMPKα1 or α2 alone or in combination were ineffective. These studies suggested a novel PPARγ-AMPK-independent mechanism of targeting GSK3β by TZD, elucidation of which might provide newer insights to improve our understanding of TRAIL-resistance.

Abstract 3322: Elucidation of the signaling pathways that mediate berberine-induced effects in cancer cells

Resistance towards standard therapeutic regimens and evasion of apoptosis are some of the hallmarks of advanced forms of cancer, which include Sorafenib-resistance in hepatocellular carcinoma (HCC), castration-resistance in prostate cancer. Development of effective therapeutic strategies that can target these resistant forms is critically needed. In an effort to understand the molecular mechanism mediating resistance in cancer, in previous studies utilizing Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistant cancer cells we showed that combination of TRAIL along with the PPARγ ligand Troglitazone (TZD) can sensitize them towards apoptosis. Using more molecular approaches these studies identified the serine/threonine protein kinase, AMP-activated protein kinase (AMPK) as a mediator of TRAIL-TZD-induced apoptosis. Since AMPK seemed to be a major mediator of this apoptosis, in the current studies we utilized the natural compound Berberine (BBR), a known activator of AMPK in combination with TRAIL. These demonstrated a significant reduction of cell viability (MTT assay) and induction of apoptosis (caspase activation) when treated with a combination of TRAIL and BBR. This apoptosis is attenuated in cells overexpressing AMPKα-dominant negative (DN), suggesting an involvement of AMPK in mediating this. To understand the downstream targets and the mechanism involved, an apoptosis PCR array analysis was performed, which suggested induction of TNFRSF10B (DR5) gene expression by BBR. In addition, knockdown of DR5 expression attenuated TRAIL-BBR-induced apoptosis, suggesting DR5 to be a potential target of BBR in this apoptotic cascade. Future studies will include determining any crosstalk of AMPK in BBR signaling to modulate DR5 pathway. Our earlier studies also demonstrated an involvement of β-catenin in mediating cancer cell resistance. Stabilizing mutations and overexpression of β-catenin has been reported in many cancers, most profoundly in HCC leading to an activation of Wnt/β-catenin signaling. To understand any role of β-catenin in TRAIL-BBR-induced apoptosis, we determined the effect of BBR on β-catenin pathway. These revealed a significant attenuation of β-catenin protein expression by BBR in various HCC cells in a time and dose-dependent manner. BBR treatment also attenuated β-catenin/TCF-induced transcriptional activity, indicating antagonism of β-catenin pathway. Our studies indicate that combination of TRAIL and AMPK activator BBR might be an effective means of antagonizing β-catenin pathway and ameliorating TRAIL resistance via DR5 in advanced forms of cancer. Citation Format: Basabi Rana, Rong Ke, Kanchan Vishnoi, Navin Viswakarma, Subhasis Das, Ajay Rana. Elucidation of the signaling pathways that mediate berberine-induced effects in cancer cells [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 3322. doi:10.1158/1538-7445.AM2017-3322

Tu1672 Involvement of p21-Activated Kinase 4 (PAK4) in Gastrin-Induced Migration of Gastric Cancer Cells

Gastro-esophageal reflux disease complicated by Barretts esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). The mechanisms whereby acid reflux may accelerate the progression from BE to EA are not fully understood. Acid and reactive oxygen species (ROS) have been reported to cause DNA damage in Barretts cells. We have previously shown that NADPH oxidase NOX5-S is responsible for acid-induced H2O2 production in Barretts cells and in EA cells. In this study we examined the role of intracellular calcium in acid-induced DNA damage in a Barretts EA cell line FLO. FLO cells were exposed acid (pH 5.0) for one hour, washed and cultured in regular culture medium for additional 24 hours. DNA damage was detected by a Comet Assay. We found that pulsed acid treatment significantly increased tail length from 1.7±0.2 to 10.1±0.6 pixels (t test, P<0.01), tail area from 51.3±7.2 to 312.7±23.5 pixels (t test, P<0.01), and tail moment from 0.7±0.1 to 3.1±0.3 (t test, P<0.01), suggesting that pulsed acid treatment increases DNA damage in FLO EA cells. In addition, acid treatment significantly increased intracellular Ca2+ concentration by 216.2±14.4% control in Fura-2/AM-loaded FLO cells, an increase which was significantly decreased by calcium-free medium plus EGTA or by thapsigargin, and almost blocked by Ca2+ free medium with EGTA and thapsigargin. Acid-induced increase in tail length, tail area, tail moment and histone H2AX phosphorylation was significantly decreased by blockade of intracellular Ca2+ increase, by NADPH oxidase inhibitor diphenylene iodonium and by knockdown of NOX5-S with NOX5 siRNA. Conversely, overexpression of NOX5-S significantly increased tail length, tail area, tail moment and histone H2AX phosphorylation. Moreover, intracellular calcium increase induced by calcium ionophore A23187 significantly increased tail length from 3.0±0.7 to 11.8±1.1 pixels (t test P<0.0001), tail area from 63.3±21.1 to 234.1±25.5 pixels (t test P<0.0001), and tail moment from 0.5±0.3 to 1.4±0.3 (t test, P<0.03), suggesting that intracellular calcium increase may cause DNA damage in FLO EA cells. We conclude that pulsed acid treatment causes DNA damage via increase of intracellular calcium and activation of NOX5-S. It is possible that in Barretts esophagus acid reflux increases intracellular calcium, activates NOX5-S and increases ROS production, which causes DNA damage, thereby contributing to the progression from BE to EA. Supported by NIH NIDDK R01 DK080703.