LncRNA SNHG3 induces EMT and sorafenib resistance by modulating the miR-128/CD151 pathway in hepatocellular carcinoma

Dysregulation of long noncoding RNAs (lncRNAs) plays important roles in carcinogenesis and tumor progression, including hepatocellular carcinoma (HCC). Small nucleolar RNA host gene 3 (SNHG3) has been considered as an lncRNA to be associated with a poor prognosis in patients with HCC. Here, we reported that SNHG3 expression was significantly higher in the highly metastatic HCC (HCCLM3) cells compared with the lowly metastatic HCC cells (Hep3B and PLC/PRF/5). Furthermore, forced expression of SNHG3 promoted cell invasion, epithelial-mesenchymal transition (EMT), and sorafenib resistance in HCC. Moreover, SNHG3 overexpression induced HCC cells EMT via miR-128/CD151 cascade activation. Clinically, our data revealed that increased SNHG3 expression is correlated with poor HCC survival outcomes and sorafenib response. These data suggest that SNHG3 may be a novel therapeutic target and a biomarker for predicting response to sorafenib treatment of HCC.     

Bufalin Reverses Resistance to Sorafenib by Inhibiting Akt Activation in Hepatocellular Carcinoma: The Role of Endoplasmic Reticulum Stress

Sorafenib is the standard first-line therapeutic treatment for patients with advanced hepatocellular carcinoma (HCC), but its use is hampered by the development of drug resistance. The activation of Akt by sorafenib is thought to be responsible for this resistance. Bufalin is the major active ingredient of the traditional Chinese medicine Chan su, which inhibits Akt activation; therefore, Chan su is currently used in the clinic to treat cancer. The present study aimed to investigate the ability of bufalin to reverse both inherent and acquired resistance to sorafenib. Bufalin synergized with sorafenib to inhibit tumor cell proliferation and induce apoptosis. This effect was at least partially due to the ability of bufalin to inhibit Akt activation by sorafenib. Moreover, the ability of bufalin to inactivate Akt depended on endoplasmic reticulum (ER) stress mediated by inositol-requiring enzyme 1 (IRE1). Silencing IRE1 with siRNA blocked the bufalin-induced Akt inactivation, but silencing eukaryotic initiation factor 2 (eIF2) or C/EBP-homologous protein (CHOP) did not have the same effect. Additionally, silencing Akt did not influence IRE1, CHOP or phosphorylated eIF2α expression. Two sorafenib-resistant HCC cell lines, which were established from human HCC HepG2 and Huh7 cells, were refractory to sorafenib-induced growth inhibition but were sensitive to bufalin. Thus, Bufalin reversed acquired resistance to sorafenib by downregulating phosphorylated Akt in an ER-stress-dependent manner via the IRE1 pathway. These findings warrant further studies to examine the utility of bufalin alone or in combination with sorafenib as a first- or second-line treatment after sorafenib failure for advanced HCC.     

Regulation of the EGFR/ErbB signalling by clathrin in response to various ligands in hepatocellular carcinoma cell lines

Membrane receptor intracellular trafficking and signalling are frequently altered in cancers. Our aim was to investigate whether clathrin‐dependent trafficking modulates signalling of the ErbB receptor family in response to amphiregulin (AR), EGF, heparin‐binding EGF‐like growth factor (HB‐EGF) and heregulin‐1β (HRG). Experiments were performed using three hepatocellular carcinoma (HCC) cell lines, Hep3B, HepG2 and PLC/PRF/5, expressing various levels of EGFR, ErbB2 and ErbB3. Inhibition of clathrin‐mediated endocytosis (CME), by down‐regulating clathrin heavy chain expression, resulted in a cell‐ and ligand‐specific pattern of phosphorylation of the ErbB receptors and their downstream effectors. Clathrin down‐regulation significantly decreased the ratio between phosphorylated EGFR (pEGFR) and total EGFR in all cell lines when stimulated with AR, EGF, HB‐EGF or HRG, except in HRG‐stimulated Hep3B cells in which pEGFR was not detectable. The ratio between phosphorylated ErbB2 and total ErbB2 was significantly decreased in clathrin down‐regulated Hep3B cells stimulated with any of the ligands, and in HRG‐stimulated PLC/PRF/5 cells. The ratio between phosphorylated ErbB3 and total ErbB3 significantly decreased in clathrin down‐regulated cell lines upon stimulation with EGF or HB‐EGF. STAT3 phosphorylation levels significantly increased in all cell lines irrespective of stimulation, while that of AKT remained unchanged, except in AR‐stimulated Hep3B and HepG2 cells in which pAKT was significantly decreased. Finally, ERK phosphorylation was insensitive to clathrin inhibition. Altogether, our observations indicate that clathrin regulation of ErbB signalling in HCC is a complex process that likely depends on the expression of ErbB family members and on the autocrine/paracrine secretion of their ligands in the tumour environment.     

Mcl-1-dependent activation of Beclin 1 mediates autophagic cell death induced by sorafenib and SC-59 in hepatocellular carcinoma cells

We investigated the molecular mechanisms underlying the effect of sorafenib and SC-59, a novel sorafenib derivative, on hepatocellular carcinoma (HCC). Sorafenib activated autophagy in a dose- and time-dependent manner in the HCC cell lines PLC5, Sk-Hep1, HepG2 and Hep3B. Sorafenib downregulated phospho-STAT3 (P-STAT3) and subsequently reduced the expression of myeloid cell leukemia-1 (Mcl-1). Inhibition of Mcl-1 by sorafenib resulted in disruption of the Beclin 1-Mcl-1 complex; however, sorafenib did not affect the amount of Beclin 1, suggesting that sorafenib treatment released Beclin 1 from binding with Mcl-1. Silencing of SHP-1 by small interference RNA (siRNA) reduced the effect of sorafenib on P-STAT3 and autophagy. Ectopic expression of Mcl-1 abolished the effect of sorafenib on autophagy. Knockdown of Beclin 1 by siRNA protected the cells from sorafenib-induced autophagy. Moreover, SC-59, a sorafenib derivative, had a more potent effect on cancer cell viability than sorafenib. SC-59 downregulated P-STAT3 and induced autophagy in all tested HCC cell lines. Furthermore, our in vivo data showed that both sorafenib and SC-59 inhibited tumor growth, downregulated P-STAT3, enhanced the activity of SHP-1 and induced autophagy in PLC5 tumors, suggesting that sorafenib and SC-59 activate autophagy in HCC. In conclusion, sorafenib and SC-59 induce autophagy in HCC through a SHP-1-STAT3-Mcl-1-Beclin 1 pathway.     

A PTEN inhibitor displays preclinical activity against hepatocarcinoma cells

Phosphatase and tensin homolog (PTEN) gene is considered a tumor suppressor gene. However, PTEN mutations rarely occur in hepatocellular carcinoma (HCC), whereas heterozygosity of PTEN, resulting in reduced PTEN expression, has been observed in 32–44% of HCC patients. In the present study, we investigated the effects of the small molecule PTEN inhibitor VO-OHpic in HCC cells. VO-OHpic inhibited cell viability, cell proliferation and colony formation, and induced senescence-associated β-galactosidase activity in Hep3B (low PTEN expression) and to a lesser extent in PLC/PRF/5 (high PTEN expression) cells, but not in PTEN-negative SNU475 cells. VO-OHpic synergistically inhibited cell viability when combined with PI3K/mTOR and RAF/MEK/ERK pathway inhibitors, but only in Hep3B cells, and significantly inhibited tumor growth in nude mice bearing xenografts of Hep3B cells. Therefore, we demonstrated for the first time that VO-OHpic inhibited cell growth and induced senescence in HCC cells with low PTEN expression, and that the combination of VO-OHpic with PI3K/mTOR and RAF/MEK/ERK inhibitors resulted in a more effective tumor cell kill. Our findings, hence, provide proof-of-principle evidence that pharmacological inhibition of PTEN may represent a promising approach for HCC therapy in a subclass of patients with a low PTEN expression.     

Increase of Bax/ Bcl-XL ratio and arrest of cell cycle by luteolin in immortalized human hepatoma cell line

Luteolin is a common constituent of many kinds of fruits and vegetables. It possesses the anti-neoplastic activities against several human cancers, but its activity against hepatocellular carcinoma (HCC) is seldom mentioned. To evaluate the activity against HCC and to provide information about the mechanism, we tested luteolin against five human hepatoma cell lines, namely HepG2, SK-Hep-1, PLC/PRF/5, Hep3B, and HA22T/VGH, with XTT assay and flow cytometry. The results showed that luteolin inhibited PLC/PRF/5, Hep3B and HA22T/VGH at a concentration of 1 microg/ml, but it needed 5 microg/ml to inhibit HepG2 and 10 microg/ml for SK-Hep1 (P <0.05). The inhibitive concentrations of 50% (IC50) of luteolin were between 7.29 microg/ml and 32.59 microg/ml, which were comparable with those of 5-FU (15.35 microg/ml to 32.84 microg/ml). The least effective cell line as affected by luteolin (SK-Hep1) was the most effective one when treating with 5-FU. The least effective cell line as affected by 5-FU (HA22T/VGH) was effectively affected by luteolin. It seemed that luteolin had some complementary activity to 5-FU against these HCC cell lines. The luteolin-treated PLC/PRF/5 cells exhibited typical changes of apoptosis with a characteristic DNA laddering pattern on gel electrophoresis. Luteolin also activated casepase-3, increased Bax protein with a concomitant decrease in Bcl-XL level. Increase in Bax/ Bcl-XL ratio and activation of caspase-3 supported the apoptotic finding on gel electrophoresis. Luteolin also induced cell cycle arrest at G0/G1 phase. We suggested that luteolin might exhibit anti-HCC activity as efficient as 5-FU by the mechanism of not only cell cycle arrest but also apoptosis.     

Targeted inhibition of transferrin-mediated iron uptake in Hep G2 hepatoma cells

We have used a model system consisting of two human hepatoma cell lines, Hep G2, representing well differentiated normal hepatocytes, and PLC/PRF/5, representing poorly differentiated malignant hepatocytes, to demonstrate that the differential presence of asialoglycoprotein receptor activity in these cell lines can be used to influence transferrin-mediated iron uptake. We based our experiments on the following facts: Hep G2 cells possess receptors that bind, internalize, and degrade galactose-terminal (asialo-)glycoproteins; PLC/PRF/5 cells have barely detectable asialoglycoprotein receptor activity; both cell lines possess active transferrin-mediated iron uptake; transferrin releases iron during acidification of intracellular vesicular compartments; primary amines, e.g. primaquine, inhibit acidification and iron release from transferrin. When added to culture medium, [55Fe]transferrin delivered 55Fe well to both cell lines. As expected, in the presence of [55Fe]transferrin, free primaquine caused a concentration-dependent decrease in 55Fe uptake in both cell lines. To create a targetable conjugate, primaquine was covalently coupled to asialofetuin to form asialofetuin-primaquine. When PLC/PRF/5 (asialoglycoprotein receptor (-)) cells were preincubated with this conjugate, transferrin-mediated 55Fe uptake was unaffected. However, transferrin-mediated 55Fe uptake by Hep G2 (asialoglycoprotein receptor (+)) cells under identical conditions was specifically decreased by 55% compared to control cells incubated without the conjugate.     

Comparison of different hepatocyte cell lines for use in a hybrid artificial liver model

Selection of a cell line suitable for a hybrid artificial liver model employing cellulose porous beads (CPBs) was investigated. Hep G2 cells grown in a culture dish exhibited appreciably higher ureogenesis and gluconeogenesis activities than those grown in CPBs. SEM observation of CPBs revealed marked difference in the distribution of attached cells from one bead to another, and showed that almost all the cell-bearing micropores were completely packed with cells. With the aim of selecting a cell line not prone to excessive aggregation and which grows moderately so as not to fill up the micropores, cells of 6 cell lines, HLE, HLF, Hep 3B, PLC/PRF/5, Huh 7 and Hep G2, were cultivated in dishes. Hep G2, HLE, and HLF increased to 5 × 10⁵ cells/cm², whereas PLC/PRF/5 grew only to 5 × 10⁴, and Hep 3B and Huh 7 up to 2 × 10⁴ cells/cm². The specific activities of ureogenesis and gluconeogenesis of Huh 7 were the highest among the lines tested - 42- and 7-fold those of Hep G2, respectively. When the 6 cell lines were grown in a submerged culture with 0.6 g/l of CPBs, Huh 7 had the lowest cell concentration of 0.54 × 10⁶ cells/ml, and the highest activities of ammonia consumption and urea and glucose production (1.38 μ mol NH₃, 99 nmol urea, and 14.5 nmol glucose/10⁶cells/h). Consequently, Huh 7 is considered to be a suitable cell line for use in the development of an artificial liver model employing porous beads. This revised version was published online in July 2006 with corrections to the Cover Date.     

Purification of alpha 1-microglobulin produced by human hepatoma cell lines. Biochemical characterization and comparison with alpha 1-microglobulin synthesized by human hepatocytes

alpha 1-Microglobulin (alpha 1m) was determined by radio-immunoassay in the supernatants of five human hepatoma cell lines. High amounts of alpha 1m were produced by PLC/PRF/5, intermediate ones by Hep G2 and Hep 3B and very low ones by Malhavu and SK Hepl. alpha 1m isolated from hepatoma cell lines PLC/PRF/5 or Hep G2 supernatants displayed the same physicochemical properties as that purified from human urines: the apparent molecular mass was 26 kDa and the pI from 5.6 to 6.4 as measured after two-dimensional polyacrylamide gel electrophoresis in denaturating conditions; for the native molecule the pI was estimated to be 4.0-4.9. Both urinary and hepatoma alpha 1m migrate as a diffuse band in the alpha zone in agarose gel at pH 8.6 in non-denaturing conditions and present a brown chromophore covalently associated with the molecule. After biosynthetic labelling with [35S]methionine, proteins extracted from hepatoma cell line PLC/PRF/5 and from isolated hepatocytes of human liver were separated by two-dimensional PAGE and transferred to a nitrocellulose membrane. alpha 1m was identified and found to be identical in both cases. However, when compared with the alpha 1m isolated from cell supernatants, less charge heterogeneity but also minor additional spots of higher molecular mass were observed.     

Plectin deficiency in liver cancer cells promotes cell migration and sensitivity to sorafenib treatment

Plectin involved in activation of kinases in cell signaling pathway and plays important role in cell morphology and migration. Plectin knockdown promotes cell migration by activating focal adhesion kinase and Rac1-GTPase activity in liver cells. Sorafenib is a multi-targeting tyrosine kinase inhibitor that improves patient survival on hepatocellular carcinoma. The aim of this study is to investigate the correlation between the expression of plectin and cell migration as well as the sensitivity of hepatoma cell lines exposing to sorafenib. Hepatoma cell lines PLC/PRF/5 and HepG2 were used to examine the level of plectin expression and cell migration in comparison with Chang liver cell line. In addition, sensitivity of the 3 cell lines to sorafenib treatment was also measured. Expression of plectin was lower in PLC/PRF/5 and HepG2 hepatoma cells than that of Chang liver cells whereas HepG2 and PLC/PRF/5 cells exhibit higher rate of cell migration in trans-well migration assay. Immunohistofluorecent staining on E-cadherin revealed the highest rate of collective cell migration in HepG2 cells and the lowest was found in Chang liver cells. Likewise, HepG2 cell line was most sensitive to sorafenib treatment and Chang liver cells exhibited the least sensitivity. The drug sensitivity to sorafenib treatment showed inverse correlation with the expression of plectin. We suggest that plectin deficiency and increased E-cadherin in hepatoma cells were associated with higher rates of cell motility, collective cell migration as well as higher drug sensitivity to sorafenib treatment.     

Hepatitis a virus infection of HBsAg-producing hepatocellular carcinomas in athymic mice

Summary Two human hepatocellular carcinoma cell lines were grown in athymic mice. Morphologically, the tumors resembled hepatocellular carcinomas. HBsAg, anti-HBs, and -fetoprotein were detected in sera of mice bearing tumors of PLC/PRF/5 or Hep 3B cells; their amounts correlated with tumor weight. Tumors of both cell lines were infected with cell culture-adapted hepatitis A virus (HAV); HAV antigen and infectious virus were recovered from infected tumors of PLC/PRF/5 and Hep 3B cells. Our results indicate that HAV-infected tumors may be useful in studying the production in vivo of hepatitis A virus antigen.     

Hepatocyte growth factor stimulates the growth and activates mitogen-activated protein kinase in human hepatoma cells

Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes and various epithelial cells. Unexpectedly, it has been reported to inhibit the growth of hepatoma cells in vitro. To clarify this phenomenon, we examined the effects of recombinant baculovirus-expressed HGF on the growth of 6 human hepatoma cell lines. The growth of Hep3B and HepG2 cells was markedly stimulated to 1.8- and 1.7-fold, respectively, PLC/PRF/5 to 1.4-fold, and SK-Hep-1 to 1.2-fold in a dose-dependent manner under HGF concentrations below 20 ng/ml. Neither HuH-7 nor HCC36 were affected. None of these cells were inhibited. All these cells expressed c-Met, the membrane receptor for HGF, and their c-Met would be activated to be phosphorylated upon addition of HGF. They also contained the ERK2 subgroup of mitogen-activated protein kinases (MAPKs). When HGF was added, their ERK2 would also be phosphorylated. The extent of ERK2 phosphorylation was partially correlated to their growth response to HGF. In conclusion, HGF could stimulate the growth of certain human hepatoma cells, probably through activation of c-Met and MAPKs.     

Involvement of the ornithine decarboxylase/polyamine system in precondition-induced cardioprotection through an interaction with PKC in rat hearts

Anion exchanger (AE) 2, belonging to the chloride–bicarbonate transporter family, has been reported to involve cell survival for hepatocellular carcinoma (HCC) cells. Our previous findings showed that AE2 gene was highly expressed in a poorly differentiated HCC cell line, HA22T/VGH. Additionally, treatment with 4,4′-diisothiocyanatostilbene-2,20-disulfonic acid (DIDS), an AE-specific inhibitor, significantly inhibited cell proliferation and induced cell apoptosis for the HA22T/VGH. To further investigate the biological functions of AE2 in human HCC, suppression of AE2 expression by the antisense oligonucleotide-AE2 (AS-AE2) was performed, and the cell viability, cell cycle regulation, and cell apoptosis for HCC cell lines were monitored. The results showed that AS-AE2 treatment could efficiently suppress the mRNA expression of AE2 for various differentiated HCC cells, including HA22T/VGH, SK-Hep-1, PLC/PRF/5, Hep3B, and HepG2. Moreover, AS-AE2 treatment significantly reduced cell viability, arrested cell cycle at sub-G1 phase, and induced cell apoptosis for the poorly differentiated HA22T/VGH, but not for other moderately or well-differentiated HCC cell lines. The findings indicated that AE2 may play an important role in the progression of HCC cells, and provide a new strategy for the development of therapeutic treatment against human HCC.     

Comprehensive kinomic study via a chemical proteomic approach reveals kinome reprogramming in hepatocellular carcinoma tissues

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Kinases are attractive therapeutic targets since they are commonly altered in cancers. Here, to identify kinases of potential therapeutic interest in HCC, a quantitative kinomic study of tumour and adjacent non-tumour liver tissues was performed using a chemical proteomics approach. In total, 124 kinases were found differentially expressed and they were distributed over all nine kinase groups. Exploration of The Cancer Genome Atlas (TCGA) data showed that the dysregulation of 45 kinases was correlated with poor prognosis in HCC patients. We then tested 11 inhibitors targeting 12 crucial protein kinases alone or in combination for their ability to inhibit cell growth in Hep3B and PLC/PRF/5 cell lines. Six inhibitors significantly reduced viability in both cell lines. Combination inhibition of polo-like kinase 1 (PLK1) and casein kinase 1 epsilon (CSNK1E) significantly induced growth arrest in both cell lines synergistically. In summary, our analysis presents the most complete view of kinome reprogramming in HCC and provides novel insight into crucial kinases in HCC and potential therapeutic targets for HCC treatment. Moreover, the identification of hundreds of differentially expressed kinases forms a rich resource for novel drug targets or diagnostic biomarker discovery. Data are available via ProteomeXchange (identifier PXD023806).     

Reduction of PKC alpha decreases cell proliferation, migration, and invasion of human malignant hepatocellular carcinoma

Protein kinase C (PKC) superfamily play key regulatory roles on the development of cancer. However, the exact role of these enzymes in human hepatocellular carcinoma (HCC) has not been well established. Using the RT-PCR and Western blotting to analyze the levels of PKC isoforms mRNA and protein in the five different differentiated hepatoma cell lines, we found that PKC alpha was highly expressed in the poor-differentiated HCC cell lines (SK-Hep-1 and HA22T/VGH) as compared with that in the well-differentiated HCC cell lines (PLC/PRF/5, Hep3B, and HepG2). When treated with PKC alpha antisense oligonucleotides (ODN), both HA22T/VGH and SK-Hep-1 cells lines showed the reduction of PKC alpha expression, as well as a deceleration in the growth rate and in the level of cyclin D1, but the increase in the levels of p53 and p21(WAF1/CIP1). Moreover, the reduction of PKC alpha expression also inhibited the migratory and invasive potential of both HA22T/VGH and SK-Hep-1 cells lines, and revealed a down-regulation of several migration/invasion-related genes (MMP-1, u-PA, u-PAR, and FAK). These phenomenon were also confirmed by DNA-based small interfering RNA (siRNA) PKC alpha and PKC alpha/beta specific inhibitor Go6976. Thus, the results indicated that PKC alpha may be associated with regulation of cell proliferation/migration/invasion in human poorly differentiated HCC cells, suggesting a role for the PKC alpha in the malignant progression of human HCC.     

Potentiating the Efficacy of Molecular Targeted Therapy for Hepatocellular Carcinoma by Inhibiting the Insulin-Like Growth Factor Pathway

Insulin-like growth factor (IGF) signaling pathway is an important regulatory mechanism of tumorigenesis and drug resistance in many cancers. The present study explored the potential synergistic effects between IGF receptor (IGFR) inhibition and other molecular targeted agents (MTA) in HCC cells. HCC cell lines (Hep3B, PLC5, and SK-Hep1) and HUVECs were tested. The MTA tested included sorafenib, sunitinib, and the IGFR kinase inhibitor NVP-AEW541. The potential synergistic antitumor effects were tested by median dose effect analysis and apoptosis assay in vitro and by xenograft models in vivo. The activity and functional significance of pertinent signaling pathways and expression of apoptosis-related proteins were measured by RNA interference and Western blotting. We found that IGF can activate IGFR and downstream AKT signaling activities in all the HCC cells tested, but the growth-stimulating effect of IGF was most prominent in Hep3B cells. NVP-AEW541 can abrogate IGF-induced activation of IGFR and AKT signaling in HCC cells. IGF can increase the resistance of HCC cells to sunitinib. The apoptosis-inducing effects of sunitinib, but not sorafenib, were enhanced when IGFR signaling activity was inhibited by NVP-AEW541 or IGFR knockdown. Chk2 kinase activation was found contributory to the synergistic anti-tumor effects between sunitinib and IGFR inhibition. Our data indicate that the apoptosis-potentiating effects of IGFR inhibition for HCC may be drug-specific. Combination therapy of IGFR inhibitors with other MTA may improve the therapeutic efficacy in HCC.     

ERK phosphorylation is dependent on cell adhesion in a subset of pediatric sarcoma cell lines

Osteosarcoma (OS) and Pax-Foxo1 fusion negative rhabdomyosarcoma (FN-RMS) are pediatric sarcomas with poor prognoses in patients with advanced disease. In both malignancies, an actin binding protein has been linked to poor prognosis. Integrin adhesion complexes (IACs) are closely coupled to actin networks and IAC-mediated signaling has been implicated in the progression of carcinomas. However, the relationship of IACs and actin cytoskeleton remodeling with cell signaling is understudied in pediatric sarcomas. Here, we tested the hypothesis that IAC dynamics affect ERK activation in OS and FN-RMS cell lines. Adhesion dependence of ERK activation differed among the OS and FN-RMS cells examined. In the OS cell lines, adhesion did not have a consistent effect on phospho-ERK (pERK). ERK phosphorylation in response to fetal calf serum or 1 ng/ml EGF was nearly as efficient in OS cell lines and one FN-RMS cell line in suspension as cells adherent to poly-l-lysine (PL) or fibronectin (FN). By contrast, adhesion to plastic, PL or FN increased ERK phosphorylation and was greater than additive with a 15 min exposure to 1 ng/ml EGF in three FN-RMS cell lines. Increases in pERK were partly dependent on FAK and PAK1/2 but independent of IAC maturation. As far as we are aware, this examination of adhesion-dependent signaling is the first in pediatric sarcomas and has led to the discovery of differences from the prevailing paradigms and differences in the degree of coupling between components in the signaling pathways among the cell lines.