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Tamoxifen (TAM) is the first-line endocrine therapy for estrogen receptor-positive (ER+) breast cancer (BC). However, acquired resistance occurs in ∼50% cases. Meanwhile, although the PI3K/AKT/mTOR pathway is a viable target for treatment of endocrine therapy-refractory patients, complex signaling feedback loops exist, which can counter the effectiveness of inhibitors of this pathway. Here, we analyzed signaling pathways and metabolism in ER+ MCF7 BC cell line and their TAM-resistant derivatives that are co-resistant to endoxifen using immunoblotting, quantitative polymerase chain reaction, and the Agilent Seahorse XF Analyzer. We found that activation of AKT and the energy-sensing kinase AMPK was increased in TAM and endoxifen-resistant cells. Furthermore, ERRα/PGC-1β and their target genes MCAD and CPT-1 were increased and regulated by AMPK, which coincided with increased fatty acid oxidation (FAO) and autophagy in TAM-resistant cells. Inhibition of AKT feedback-activates AMPK and ERRα/PGC-1β-MCAD/CPT-1 with a consequent increase in FAO and autophagy that counters the therapeutic effect of endoxifen and AKT inhibitors. Therefore, our results indicate increased activation of AKT and AMPK with metabolic reprogramming and increased autophagy in TAM-resistant cells. Simultaneous inhibition of AKT and FAO/autophagy is necessary to fully sensitize resistant cells to endoxifen.

Wild-type p53 is a stress-responsive transcription factor and potent tumor suppressor. P53 activates or represses genes involved in cell cycle progression or apoptosis in order to arrest the cell cycle or induce cell death. Transcription repression by p53 is indirect and requires repressive members of the RB-family (RB1, RBL1, RBL2) and formation of repressor complexes of RB1-E2F and RBL1/RBL2-DREAM. Many aurora kinase A/B (AURKA/B) pathway genes are repressed in a p53-DREAM-dependent manner. We found heightened expression of RBL2 and reduced expression of AURKA/B pathway genes is associated with improved outcomes in p53 wild-type but not p53 mutant non-small cell lung cancer (NSCLC) patients. Knockdown of p53, RBL2, or the DREAM component LIN37 increased AURKA/B pathway gene expression and reduced paclitaxel and radiation toxicity in NSCLC cells. In contrast, pharmacologic inhibition of AURKA/B or knockdown of AURKA/B pathway components increased paclitaxel and IR sensitivity. The results support a model in which p53-RBL2-DREAM-mediated repression of the AURKA/B pathway contributes to tumor suppression, improved tumor therapy responses, and better outcomes in p53 wild-type NSCLCs.

Platinum-based drugs such as cisplatin (CP) are the first-line chemotherapy for non-small-cell lung carcinoma (NSCLC). Unfortunately, NSCLC has a low response rate to CP and acquired resistance always occurs. Histone methylation regulates chromatin structure and is implicated in DNA repair. We hypothesize histone methylation regulators are involved in CP resistance. We therefore screened gene expression of known histone methyltransferases and demethylases in three NSCLC cell lines with or without acquired resistance to CP. JMJD2s are a family of histone demethylases that remove tri-methyl groups from H3K9 and H3K36. We found expression of several JMJD2 family genes upregulated in CP-resistant cells, with JMJD2B expression being upregulated in all three CP-resistant NSCLC cell lines. Further analysis showed increased JMJD2 protein expression coincided with decreased H3K9me3 and H3K36me3. Chemical inhibitors of JMJD2-family proteins increased H3K9me3 and H3K36me3 levels and sensitized resistant cells to CP. Mechanistic studies showed that JMJD2 inhibition decreased chromatin association of ATR and Chk1 and inhibited the ATR-Chk1 replication checkpoint. Our results reveal that JMJD2 demethylases are potential therapeutic targets to overcome CP resistance in NSCLC.

For several decades there has been accumulating evidence implicating type I interferons (IFNs) as key elements of the immune response. Therapeutic approaches incorporating different recombinant type I IFN proteins have been successfully employed to treat a diverse group of diseases with significant and positive outcomes. The biological activities of type I IFNs are consequences of signaling events occurring in the cytoplasm and nucleus of cells. Biochemical events involving JAK/STAT proteins that control transcriptional activation of IFN-stimulated genes (ISGs) were the first to be identified and are referred to as “canonical” signaling. Subsequent identification of JAK/STAT-independent signaling pathways, critical for ISG transcription and/or mRNA translation, are denoted as “non-canonical” or “non-classical” pathways. In this review, we summarize these signaling cascades and discuss recent developments in the field, specifically as they relate to the biological and clinical implications of engagement of both canonical and non-canonical pathways.

Activated p53 can promote apoptosis or cell cycle arrest. Differences in energy metabolism can influence cell fate in response to activated p53. Nutlin-3a is a preclinical drug and small molecule activator of p53. Alpha-ketoglutarate (αKG) levels were reduced in cells sensitive to Nutlin-3a-induced apoptosis and increased in cells resistant to this apoptosis. Add-back of a cell-permeable αKG analog (DMKG) rescued cells from apoptosis in response to Nutlin-3a. OGDH is a component of the αKGDH complex that converts αKG to succinate. OGDH knockdown increased endogenous αKG levels and also rescued cells from Nutlin-3a-induced apoptosis. We previously showed reduced autophagy and ATG gene expression contributes to Nutlin-3a-induced apoptosis. DMKG and OGDH knockdown restored autophagy and ATG gene expression in Nutlin-3a-treated cells. These studies indicate αKG levels, regulated by p53 and OGDH, determine autophagy and apoptosis in response to Nutlin-3a.

Autophagy promotes cancer cell survival in response to p53 activation by the anticancer agent Nutlin-3a (Nutlin). We reported previously that Nutlin kills MDM2-amplified cancer cells and that this killing is associated with an inhibition of glucose metabolism, reduced α-ketoglutarate (α-KG) levels, and reduced autophagy. In the current report, using SJSA1, U2OS, A549, and MHM cells, we found that Nutlin alters histone methylation in an MDM2 proto-oncogene–dependent manner and that this, in turn, regulates autophagy-related gene (ATG) expression and cell death. In MDM2-amplified cells, Nutlin increased histone (H) 3 lysine (K) 9 and K36 trimethylation (me3) coincident with reduced autophagy and increased apoptosis. Blocking histone methylation restored autophagy and rescued these cells from Nutlin-induced killing. In MDM2-nonamplified cells, H3K9me3 and H3K36me3 levels were either reduced or not changed by the Nutlin treatment, and this coincided with increased autophagy and cell survival. Blocking histone demethylation reduced autophagy and sensitized these cells to Nutlin-induced killing. Further experiments suggested that MDM2 amplification increases histone methylation in Nutlin-treated cells by causing depletion of the histone demethylase Jumonji domain-containing protein 2B (JMJD2B). Finally, JMJD2B knockdown or inhibition increased H3K9/K36me3 levels, decreased ATG gene expression and autophagy, and sensitized MDM2-nonamplified cells to apoptosis. Together, these results support a model in which MDM2- and JMJD2B-regulated histone methylation levels modulate ATG gene expression, autophagy, and cell fate in response to the MDM2 antagonist Nutlin-3a.

// Batzaya Davaadelger 1 , Lei Duan 1 , Ricardo E. Perez 1 , Steven Gitelis 2 , Carl G. Maki 1 1 Department of Anatomy and Cell Biology, Rush University Medical Center, Chicago, IL, USA 2 Section of Orthopedic Oncology, Department of Orthopedic Surgery, Rush University, Medical Center, Chicago, IL, USA Correspondence to: Carl G. Maki, e-mail: Carl_Maki@rush.edu Keywords: p53, p27, IGF-1R/AKT/mTORC1, cisplatin Received: January 08, 2016 Accepted: March 18, 2016 Published: March 30, 2016 ABSTRACT The insulin-like growth factor-1 receptor (IGF-1R) signaling pathway is aberrantly activated in multiple cancers and can promote proliferation and chemotherapy resistance. Multiple IGF-1R inhibitors have been developed as potential therapeutics. However, these inhibitors have failed to increase patient survival when given alone or in combination with chemotherapy agents. The reason(s) for the disappointing clinical effect of these inhibitors is not fully understood. Cisplatin (CP) activated the IGF-1R/AKT/mTORC1 pathway and stabilized p53 in osteosarcoma (OS) cells. p53 knockdown reduced IGF-1R/AKT/mTORC1 activation by CP, and IGF-1R inhibition reduced the accumulation of p53. These data demonstrate positive crosstalk between p53 and the IGF-1R/AKT/mTORC1 pathway in response to CP. Further studies showed the effect of IGF-1R inhibition on CP response is dependent on p53 status. In p53 wild-type cells treated with CP, IGF-1R inhibition increased p53s apoptotic function but reduced p53-dependent senescence, and had no effect on long term survival. In contrast, in p53-null/knockdown cells, IGF-1R inhibition reduced apoptosis in response to CP and increased long term survival. These effects were due to p27 since IGF-1R inhibition stabilized p27 in CP-treated cells, and p27 depletion restored apoptosis and reduced long term survival. Together, the results demonstrate 1) p53 expression determines the effect of IGF-1R inhibition on cancer cell CP response, and 2) crosstalk between the IGF-1R/AKT/mTORC1 pathway and p53 and p27 can reduce cancer cell responsiveness to chemotherapy and may ultimately limit the effectiveness of IGF-1R pathway inhibitors in the clinic.

MDM2 antagonists stabilize and activate wild-type p53, and histone methyltransferase (HMT) inhibitors reduce methylation on histone lysines and arginines. Both MDM2 antagonists and HMT inhibitors are being developed as cancer therapeutics. Wild-type p53 expressing HCT116 colon cancer cells were resistant to apoptosis in response to the MDM2 antagonist Nutlin-3a. However, co-treatment with the HMT inhibitor DZNep sensitized the cells to Nutlin-3a-induced apoptosis. This sensitization resulted from reduced activity of the Bcl-2 gene promoter and a reduction in Bcl-2 mRNA and protein. Surprisingly, DZNep reduced Bcl-2 expression in other colon cancer cell lines (RKO, SW48, and LoVo) but failed to sensitize them to Nutlin-3a. We found these cell lines express elevated levels of Bcl-2 or other Bcl-2-family proteins, including Bcl-xL, Mcl-1, and Bcl-w. Knockdown of Mcl-1 and/or treatment with specific or pan Bcl-2-family inhibitors (BH3 mimetics) sensitized RKO, SW48, and LoVo cells to apoptosis by Nutlin-3a. The results demonstrate 1) DZNep represses the Bcl-2 gene promoter and affects apoptosis sensitivity by reducing Bcl-2 protein expression, and 2) elevated expression of pro-survival Bcl-2 family members protects colon cancer cells from Nutlin-3a-induced apoptosis. Targeting Bcl-2 proteins via DZNep or BH3 mimetics could increase the therapeutic potential of MDM2-antagonists like Nutlin-3a in colon cancer.

The onset and degree of injury occurring in animals that develop hyperoxic acute lung injury (HALI) is dependent on age at exposure, suggesting that developmentally regulated pathways/factors must underlie initiation of the epithelial injury and subsequent repair. Type II TGFβ receptor interacting protein‐1 (TRIP‐1) is a negative regulator of TGFβ signaling, which we have previously shown is a developmentally regulated protein with modulatory effects on epithelial‐fibroblastic signaling. The aim of this study was to assess if type II alveolar epithelial cells overexpressing TRIP‐1 are protected against hyperoxia‐induced epithelial injury, and in turn HALI. Rat lung epithelial cells (RLE) overexpressing TRIP‐1 or LacZ were exposed to 85% oxygen for 4 days. A surfactant protein C (SPC)‐driven TRIP‐1 overexpression mouse (TRIP‐1 AECTg+ ) was generated and exposed to hyperoxia (>95% for 4 days) at 4 weeks of age to assess the effects TRIP‐1 overexpression has on HALI. RLE overexpressing TRIP‐1 resisted hyperoxia‐induced apoptosis. Mice overexpressing TRIP‐1 in their lung type II alveolar epithelial cells (TRIP‐1 AECTg+ ) showed normal lung development, increased phospho‐AKT level and E‐cadherin, along with resistance to HALI, as evidence by less TGFβ activation, apoptosis, alveolar macrophage influx, KC expression. Taken together, these findings point to existence of a TRIP‐1 mediated molecular pathway affording protection against epithelial/acute lung injury.

The tumor suppressor p53 regulates downstream targets that determine cell fate. Canonical p53 functions include inducing apoptosis, growth arrest, and senescence. Non-canonical p53 functions include its ability to promote or inhibit autophagy and its ability to regulate metabolism. The extent to which autophagy and/or metabolic regulation determines cell fate by p53 is unclear. To address this, we compared cells resistant or sensitive to apoptosis by the p53 activator Nutlin-3a. In resistant cells, glycolysis was maintained upon Nutlin-3a treatment, and activated p53 promoted prosurvival autophagy. In contrast, in apoptosis sensitive cells activated p53 increased superoxide levels and inhibited glycolysis through repression of glycolytic pathway genes. Glycolysis inhibition and increased superoxide inhibited autophagy by repressing ATG genes essential for autophagic vesicle maturation. Inhibiting glycolysis increased superoxide and blocked autophagy in apoptosis-resistant cells, causing p62-dependent caspase-8 activation. Finally, treatment with 2-DG or the autophagy inhibitors chloroquine or bafilomycin A1 sensitized resistant cells to Nutlin-3a-induced apoptosis. Together, these findings reveal novel links between glycolysis and autophagy that determine apoptosis-sensitivity in response to p53. Specifically, the findings indicate 1) that glycolysis plays an essential role in autophagy by limiting superoxide levels and maintaining expression of ATG genes required for autophagic vesicle maturation, 2) that p53 can promote or inhibit autophagy depending on the status of glycolysis, and 3) that inhibiting protective autophagy can expand the breadth of cells susceptible to Nutlin-3a induced apoptosis.

Nutlin-3a is a MDM2 antagonist and preclinical drug that activates p53. Cells with MDM2 gene amplification are especially prone to Nutlin-3a-induced apoptosis, though the basis for this is unclear. Glucose metabolism can inhibit apoptosis in response to Nutlin-3a through mechanisms that are incompletely understood. Glucose metabolism through the pentose phosphate pathway (PPP) produces NADPH that can protect cells from potentially lethal reactive oxygen species (ROS). We compared apoptosis and glucose metabolism in cancer cells with and without MDM2 gene amplification treated with Nutlin-3a. Apoptosis in MDM2-amplified cells was associated with a reduction in glycolysis and the PPP, reduced NADPH, increased ROS, and depletion of the transcription factor SP1, which normally promotes PPP gene expression. In contrast, glycolysis and the PPP were maintained or increased in MDM2 non-amplified cells treated with Nutlin-3a. This was dependent on p53-mediated AKT activation and was associated with maintenance of SP1 and continued expression of PPP genes. Knockdown or inhibition of AKT, SP1, or the PPP sensitized MDM2-non-amplified cells to apoptosis. The data indicate that p53 promotes AKT and SP1-dependent activation of the PPP that protects cells from Nutlin-3a-induced apoptosis. These findings provide insight into how glucose metabolism reduces Nutlin-3a-induced apoptosis, and also provide a mechanism for the heightened sensitivity of MDM2-amplified cells to apoptosis in response to Nutlin-3a.

The effectiveness of DNA damaging chemotherapy drugs can be limited by activation of survival signaling pathways and cell cycle checkpoints that allow DNA repair. Targeting survival pathways and inhibiting cell cycle checkpoints may increase chemotherapy-induced cancer cell killing. AKT and Chk1 are survival and cell cycle checkpoint kinases, respectively, that can be activated by DNA damage. Cisplatin (CP) is a standard chemotherapy agent for osteosarcoma (OS). CP induced apoptosis to varying extents and activated AKT and Chk1 in multiple p53 wild-type and p53-null OS cell lines. A Chk1 inhibitor increased CP-induced apoptosis in all OS cell lines regardless of p53 status. In contrast, an AKT inhibitor increased CP-induced apoptosis only in p53 wild-type OS cells, but not p53 nulll cells. The increased apoptosis in p53 wild-type cells was coincident with decreased p53 protein levels, but increased expression of p53-responsive apoptotic genes Noxa and PUMA. Further studies revealed the inability of AKT inhibitor to CP-sensitize p53-null OS cells resulted from 2 things: 1) AKT inhibition stabilized/maintained p27 levels in CP-treated cells, which then mediated a protective G1-phase cell cycle arrest, 2) AKT inhibition increased the levels of activated Chk1. Finally, schedule dependent inhibition of AKT and Chk1 evaded the protective G1 arrest mediated by p27 and maximized CP-induced OS cell killing. These data demonstrate AKT and Chk1 activation promote survival in CP-treated OS cells, and that strategic, scheduled targeting of AKT and Chk1 can maximize OS cell killing by CP.

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT)/mammalian target of rapamycin (mTOR) pathway conveys signals from receptor tyrosine kinases (RTKs) to regulate cell metabolism, proliferation, survival, and motility. Previously we found that prolylcarboxypeptidase (PRCP) regulate proliferation and survival in breast cancer cells. In this study, we found that PRCP and the related family member prolylendopeptidase (PREP) are essential for proliferation and survival of pancreatic cancer cells. Depletion/inhibition of PRCP and PREP-induced serine phosphorylation and degradation of IRS-1, leading to inactivation of the cellular PI3K and AKT. Notably, depletion/inhibition of PRCP/PREP destabilized IRS-1 in the cells treated with rapamycin, blocking the feedback activation PI3K/AKT. Consequently, inhibition of PRCP/PREP enhanced rapamycin-induced cytotoxicity. Thus, we have identified PRCP and PREP as a stabilizer of IRS-1 which is critical for PI3K/AKT/mTOR signaling in pancreatic cancer cells.

p53 gene mutations are among the most common alterations in cancer. In most cases, missense mutations in one TP53 allele are followed by loss-of-heterozygosity (LOH), so tumors express only mutant p53. TP53 mutations and LOH have been linked, in many cases, with poor therapy response and worse outcome. Despite this, remarkably little is known about how TP53 point mutations are acquired, how LOH occurs, or the cells involved. Nutlin-3a occupies the p53-binding site in MDM2 and blocks p53-MDM2 interaction, resulting in the stabilization and activation of p53 and subsequent growth arrest or apoptosis. We leveraged the powerful growth inhibitory activity of Nutlin-3a to select p53-mutated cells and examined how TP53 mutations arise and how the remaining wild-type allele is lost or inactivated. Mismatch repair (MMR)-deficient colorectal cancer cells formed heterozygote (p53 wild-type/mutant) colonies when cultured in low doses of Nutlin-3a, whereas MMR-corrected counterparts did not. Placing these heterozygotes in higher Nutlin-3a doses selected clones in which the remaining wild-type TP53 was silenced. Our data suggest silencing occurred through a novel mechanism that does not involve DNA methylation, histone methylation, or histone deacetylation. These data indicate MMR deficiency in colorectal cancer can give rise to initiating TP53 mutations and that TP53 silencing occurs via a copy-neutral mechanism. Moreover, the data highlight the use of MDM2 antagonists as tools to study mechanisms of TP53 mutation acquisition and wild-type allele loss or silencing in cells with defined genetic backgrounds.

Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells undergo conversion to a mesenchymal phenotype contributing to wound repair by fibrosis and to cancer cell acquisition of invasive ability. Recently, we showed that type II TGF-β receptor interacting protein-1 (TRIP-1), a protein identified as a phosphorylation target of the TGF-β type II receptor kinase and as a functional component of eukaryotic translation initiator factor 3 (eiF3) multiprotein complex, is a novel modulator of fibroblast collagen contraction, an important step in wound repair stimulated by TGF-β1 action. TGF-β1 drives EMT, but it is not known whether TRIP-1 expression influences EMT induction. To investigate whether TRIP-1 plays a role in EMT induction we studied the effect of downregulating TRIP-1 expression in the well-characterized A549 model of TGF-β1 induction of EMT. Here we report that short hairpin RNA (shRNA)-mediated depletion of TRIP-1 gene transcripts in A549 cells promotes EMT as assessed by changes in phenotypic markers, morphology, and migrative ability. Knockdown of TRIP-1 dramatically increased A549 responsiveness to TGF-β1 induction of EMT. Mechanistically, a pathway involving increased TGF-β type II receptor level, enhanced Smad3 phosphorylation, and the transcription factor SLUG is implicated. Altogether, the findings point to regulation of endogenous TRIP-1 protein expression as a potential strategy to target EMT, and related invasive behavior, in cancer cells.

Tumor-derived mutant forms of p53 compromise its DNA binding, transcriptional, and growth regulatory activity in a manner that is dependent upon the cell-type and the type of mutation. Given the high frequency of p53 mutations in human tumors, reactivation of the p53 pathway has been widely proposed as beneficial for cancer therapy. In support of this possibility p53 mutants possess a certain degree of conformational flexibility that allows for re-induction of function by a number of structurally different artificial compounds or by short peptides. This raises the question of whether physiological pathways for p53 mutant reactivation also exist and can be exploited therapeutically. The activity of wild-type p53 is modulated by various acetyl-transferases and deacetylases, but whether acetylation influences signaling by p53 mutant is still unknown. Here, we show that the PCAF acetyl-transferase is down-regulated in tumors harboring p53 mutants, where its re-expression leads to p53 acetylation and to cell death. Furthermore, acetylation restores the DNA-binding ability of p53 mutants in vitro and expression of PCAF, or treatment with deacetylase inhibitors, promotes their binding to p53-regulated promoters and transcriptional activity in vivo. These data suggest that PCAF-mediated acetylation rescues activity of at least a set of p53 mutations. Therefore, we propose that dis-regulation of PCAF activity is a pre-requisite for p53 mutant loss of function and for the oncogenic potential acquired by neoplastic cells expressing these proteins. Our findings offer a new rationale for therapeutic targeting of PCAF activity in tumors harboring oncogenic versions of p53.

We have shown previously that T1α/podoplanin is required for capillary tube formation by human lung microvascular lymphatic endothelial cells (HMVEC-LLy) and that cells with decreased podoplanin expression fail to properly activate the small GTPase RhoA shortly after the beginning of the lymphangiogenic process. The objective of this study was to determine whether podoplanin regulates HMVEC-LLy migration and whether this regulation is via modulation of small GTPase activation. In analysis of scratch wound assays, we found that small interfering RNA (siRNA) depletion of podoplanin expression in HMVEC-LLy inhibits VEGF-induced microtubule-organizing center (MTOC) and Golgi polarization and causes a dramatic reduction in directional migration compared with control siRNA-transfected cells. In addition, a striking redistribution of cortical actin to fiber networks across the cell body is observed in these cells, and, remarkably, it returns to control levels if the cells are cotransfected with a dominant-negative mutant of Cdc42. Moreover, cotransfection of a dominant-negative construct of Cdc42 into podoplanin knockdown HMVEC-LLy completely abrogated the effect of podoplanin deficiency, rescuing MTOC and Golgi polarization and cell migration to control level. Importantly, expression of constitutively active Cdc42 construct, like podoplanin knockdown, decreased RhoA-GTP level in HMVEC-LLy, demonstrating cross talk between both GTPases. Taken together, the results indicate that polarized migration of lymphatic endothelial cells in response to VEGF is mediated via a pathway of podoplanin regulation of small GTPase activities, in particular Cdc42.

Supplemental oxygen therapy (hyperoxia) in preterm babies with respiratory stress is associated with lung injury and the development of bronchopulmonary dysplasia. Endoplasmic reticulum (ER) homeostasis plays critical roles in maintaining cellular functions such as protein synthesis, folding, and secretion. Interruption of ER homeostasis causes ER stress and triggers the unfolded protein response, which can lead to apoptosis in persistently stressed cells. ERp57 is an ER protein and is associated with calreticulin and calnexin in protein glycosylation. In this study, we found hyperoxia downregulated ERp57 in neonatal rat lungs and cultured human endothelial cells. Transient transfection of ERp57 small interfering RNA significantly knocked down ERp57 expression and reduced hyperoxia- or tunicamycin-induced apoptosis in human endothelial cells. Apoptosis was decreased from 26.8 to 9.9% in hyperoxia-exposed cells and from 37.8 to 5.0% in tunicamycin-treated cells. The activation of caspase-3 induced by hyperoxia or tunicamycin was diminished and immunoglobulin heavy chain-binding protein/glucose-regulated protein 78-kDa (BiP/GRP78) induction was increased in ERp57 knockdown cells. Overexpression of ERp57 exacerbated hyperoxia- or tunicamycin-induced apoptosis in human endothelial cells. Apoptosis was increased from 10.1 to 14.3% in hyperoxia-exposed cells and from 14.0 to 21.2% in tunicamycin-treated cells. Overexpression of ERp57 also augmented tunicamycin-induced caspase-3 activation and reduced BiP/GRP78 induction. Our results demonstrate that ERp57 can regulate apoptosis in human endothelial cells. It appears that knockdown of ERp57 confers cellular protection against hyperoxia- or tunicamycin-induced apoptosis by inhibition of caspase-3 activation and stimulation of BiP/GRP78 induction.

Acute lung injury involving extremely immature lungs often heals without excessive fibrosis unlike later in gestation and in adults. Several factors may be involved, but fibroblast contraction of collagen has been linked to the level of wound fibrosis. To assess whether human lung fibroblasts of fetal versus adult origin differ in ability to contract collagen and define the molecular underpinnings, we performed three-dimensional collagen contraction assay, analyzed their differential mRNA profile, specifically for transforming growth factor-beta (TGF-beta) signaling pathway and extracellular matrix components, studied the cell response to TGF-beta in culture, and used two-dimensional gel electrophoresis followed by mass spectrometry to identify differences in their overall proteomes. Human lung fetal fibroblasts contracted the collagen matrix less than the adults. Smooth muscle actin expression did not differ. TGF-beta stimulation resulted in greater Smad3 phosphorylation in fetal compared with adults. mRNA and proteomic profiling reveal a number of TGF-beta pathways, ECM components, and cytoskeletal regulatory molecules are differentially expressed between the cell types. Of note is TGF-beta receptor interacting protein 1 (TRIP-1), which we show inhibits fibroblast collagen contraction and is higher in fetal than adult fibroblasts. We conclude that human lung fetal fibroblasts are less able to contract collagen than adult lung fibroblasts. The diminished ability is not due to impediment of Smad3 activation but rather, at least in part, due to their higher level of TRIP-1 expression. TRIP-1 is a novel modulator of fibroblast collagen contraction.

Oxygen toxicity or hyperoxia is one of the major contributing factors in the development of bronchopulmonary dysplasia. Heat shock protein 27 (Hsp27) is an important chaperone protein in the postnatal lung development. However, the role of Hsp27 in lung epithelial cells during hyperoxia is unclear. Our studies by cDNA array and immunohistochemistry revealed that hyperoxia decreased Hsp27 expression in newborn rat lungs. Western blot showed that hyperoxic treatment significantly decreased Hsp27 protein expression in cultured human lung epithelial cells (A549). The expression of Hsp27 was decreased approximately twofold after 24-h and threefold after 48- and 72-h hyperoxic exposure compared with that of the A549 cells exposed to normoxia (p < 0.05, n = 3). Knockdown of Hsp27 expression by siRNA resulted in more apoptotic cell death in A549 cells. Overexpression of Hsp27 reduced hyperoxia-induced apoptotic cell death to 9.2% in Hsp27 overexpressing A549 cells from 12.6% in control A549 cells after 72-h hyperoxic exposure (p < 0.01, n = 8-9). Overexpression of Hsp27 also diminished hyperoxia-induced caspase-9 activation in A549 cells. Our results demonstrated that hyperoxia decreased Hsp27 expression in newborn rat lung and cultured human lung epithelial cells. Overexpression of Hsp27 could reduce hyperoxia-induced apoptosis in cultured human lung epithelial cells.

The lymphatic vasculature functions to maintain tissue perfusion homeostasis. Defects in its formation or disruption of the vessels result in lymphedema, the effective treatment of which is hampered by limited understanding of factors regulating lymph vessel formation. Mice lacking T1α/podoplanin, a lymphatic endothelial cell transmembrane protein, have malformed lymphatic vasculature with lymphedema at birth, but the molecular mechanism for this phenotype is unknown. Here, we show, using primary human lung microvascular lymphatic endothelial cells (HMVEC-LLy), that small interfering RNA-mediated silence of podoplanin gene expression has the dramatic effect of blocking capillary tube formation in Matrigel. In addition, localization of phosphorylated ezrin/radixin/moesin proteins to plasma membrane extensions, an early event in the capillary morphogenic program in lymphatic endothelial cells, is impaired. We find that cells with decreased podoplanin expression fail to properly activate the small GTPase RhoA early (by 30 min) after plating on Matrigel, and Rac1 shows a delay in its activation. Further indication that podoplanin action is linked to RhoA activation is that use of a cell-permeable inhibitor of Rho inhibited lymphatic endothelial capillary tube formation in the same manner as did podoplanin gene silencing, which was not mimicked by treatment with a Rac1 inhibitor. These data clearly demonstrate that early activation of RhoA in the lymphangiogenic process, which is required for the successful establishment of the capillary network, is dependent on podoplanin expression. To our knowledge, this is the first time that a mechanism has been suggested to explain the role of podoplanin in lymphangiogenesis.

Hyperoxia is one of the major contributors to the development of bronchopulmonary dysplasia (BPD), a chronic lung disease in premature infants. Emerging evidence suggests that the arrested lung development of BPD is associated with pulmonary endothelial cell death and vascular dysfunction resulting from hyperoxia-induced lung injury. A better understanding of the mechanism of hyperoxia-induced endothelial cell death will provide critical information for the pathogenesis and therapeutic development of BPD. Epidermal growth factor-like domain 7 (EGFL7) is a protein secreted from endothelial cells. It plays an important role in vascular tubulogenesis. In the present study, we found that Egfl7 gene expression was significantly decreased in the neonatal rat lungs after hyperoxic exposure. The Egfl7 expression was returned to near normal level 2 wk after discounting oxygen exposure during recovery period. In cultured human endothelial cells, hyperoxia also significantly reduced Egfl7 expression. These observations suggest that diminished levels of Egfl7 expression might be associated with hyperoxia-induced endothelial cell death and lung injury. When we overexpressed human Egfl7 ( hEgfl7) in EA.hy926 human endothelial cell line, we found that hEgfl7 overexpression could partially block cytochrome c release from mitochondria and decrease caspase-3 activation. Further Western blotting analyses showed that hEgfl7 overexpression could reduce expression of a proapoptotic protein, Bax, and increase expression of an antiapoptotic protein, Bcl-xL. Theses findings indicate that hEGFL7 may protect endothelial cell from hyperoxia-induced apoptosis by inhibition of mitochondria-dependent apoptosis pathway.

Cisplatin is a platinum-based drug that is used for the treatment of a wide-variety of primary human cancers. However, the therapeutic efficacy of cisplatin is often limited by intrinsic or acquired drug resistance. An important goal, therefore, is to identify mechanisms that lead to cisplatin resistance in cancer, and then use this information to more effectively target resistant cells. Cisplatin-resistant clones of the HCT116 cell line underwent a prolonged G2 arrest after cisplatin treatment while sensitive clones did not. The staurosporine analog UCN-01 abrogated this G2 arrest and sensitized the resistant clones to cisplatin. At later time points, 4N arrested cells assumed a tetraploid G1 state that was characterized by depletion of Cyclin A, Cyclin B, and CDC2, and increased expression of p53 and p21, in 4N cells. siRNA-mediated knockdown of p21 abrogated the tetraploid G1 arrest and induced killing that was dependent on p53. The results identify two targetable 4N arrests that can contribute to cisplatin resistance: First, a prolonged G2 arrest that can be targeted by UCN-01, and second, a tetraploid G1 arrest that can be targeted by siRNA against p21.

Background Myofibroblasts are the critical effector cells in the pathogenesis of pulmonary fibrosis which carries a high degree of morbidity and mortality. We have previously identified Type II TGFβ receptor interacting protein 1 (TRIP-1), through proteomic analysis, as a key regulator of collagen contraction in primary human lung fibroblasts—a functional characteristic of myofibroblasts, and the last, but critical step in the process of fibrosis. However, whether or not TRIP-1 modulates fibroblast trans-differentiation to myofibroblasts is not known. Methods TRIP-1 expression was altered in primary human lung fibroblasts by siRNA and plasmid transfection. Transfected fibroblasts were then analyzed for myofibroblast features and function such as α-SMA expression, collagen contraction ability, and resistance to apoptosis. Results The down-regulation of TRIP-1 expression in primary human lung fibroblasts induces α-SMA expression and enhances resistance to apoptosis and collagen contraction ability. In contrast, TRIP-1 over-expression inhibits α-SMA expression. Remarkably, the effects of the loss of TRIP-1 are not abrogated by blockage of TGFβ ligand activation of the Smad3 pathway or by Smad3 knockdown. Rather, a TRIP-1 mediated enhancement of AKT phosphorylation is the implicated pathway. In TRIP-1 knockdown fibroblasts, AKT inhibition prevents α-SMA induction, and transfection with a constitutively active AKT construct drives collagen contraction and decreases apoptosis. Conclusions TRIP-1 regulates fibroblast acquisition of phenotype and function associated with myofibroblasts. The importance of this finding is it suggests TRIP-1 expression could be a potential target in therapeutic strategy aimed against pathological fibrosis.