Male germ cell-Associated Kinase (MAK), a direct transcriptional target of androgen receptor (AR), is a coactivator of AR. In this study, we determined the activating mechanism of MAK and identified a previously unknown AR-independent role of MAK in mitosis. We found that MAK kinase activity requires dual phosphorylation of the conserved TDY motif and that the phosphorylation is dynamic during cell cycle. MAK associates with CDH1 (FZR1, fizzy/cell division cycle 20 related 1) and phosphorylates CDH1 at sites phosphorylated by CDK. When MAK is overexpressed, the binding of CDH1 to Anaphase Promoting Complex/Cyclosome decreased, resulting in an attenuation of APC/C ubiquitin ligase activity and the consequential stabilization of the CDH1 targets such as Aurora kinase A and PLK1. As such, overexpression of MAK leads to mitotic defects such as centrosome amplification and lagging chromosomes. Our immunohistochemistry result showed that MAK is overexpressed in prostate tumor tissues, suggesting a role of MAK in prostate carcinogenesis. Taken with our previous results, our data implicate MAK in both AR activation and chromosomal instability, acting in both early and late prostate cancer (PCA) development.

Within solid tumor microenvironments, lactic acidosis and hypoxia each have powerful effects on cancer pathophysiology. However, the influence that these processes exert on each other is unknown. Here we report that a significant portion of the transcriptional response to hypoxia elicited in cancer cells is abolished by simultaneous exposure to lactic acidosis. In particular, lactic acidosis abolished stabilization of HIF-1α protein which occurs normally under hypoxic conditions. In contrast, lactic acidosis strongly synergized with hypoxia to activate the unfolded protein response (UPR) and an inflammatory response, displaying a strong similarity to ATF4-driven amino acid deprivation responses (AAR). In certain breast tumors and breast tumor cells examined, an integrative analysis of gene expression and array CGH data revealed DNA copy number alterations at the ATF4 locus, an important activator of the UPR/AAR pathway. In this setting, varying ATF4 levels influenced the survival of cells after exposure to hypoxia and lactic acidosis. Our findings reveal that the condition of lactic acidosis present in solid tumors inhibits canonical hypoxia responses and activates UPR and inflammation responses. Further, they suggest that ATF4 status may be a critical determinant of the ability of cancer cells to adapt to oxygen and acidity fluctuations in the tumor microenvironment, perhaps linking short-term transcriptional responses to long-term selection for copy number alterations in cancer cells.

It is known that Ras mutations, together with loss of PKC, are apoptotic in various types of mammalian cells. The mechanism of how aberrant Ras transmits this apoptotic signaling remains unclear. Using three V12-Ha-ras loop mutants that preferentially bind to and activate one of Ras effectors, we tested the role of Ras downstream pathways in the induction of apoptosis in rat lung epithelia, human lung or prostate cancer cells. After PKC inhibition, the activation of PI3K/Akt renders the susceptibility of cells to apoptosis. We also demonstrate that the amount of ROS is moderately increased in the cells ectopically expressing V12C40 and dramatically elevated by suppression of PKC, which leads to apoptosis through the activation of UPR. Thus, our study suggests that after PKC abrogation, PI3K functions downstream of Ras to perturb the state of cellular redox and signals to ER stress-regulated apoptotic machinery.

Background

Gastric cancer is a common and highly lethal malignancy in the world, but its pathogenesis remains elusive. In this study, we focus on the biological functions of CDK-associated Cullin1 (CAC1), a novel gene of the cullin family, in gastric cancer, which may help us to further understand the origin of this malignancy.

Methods

The AGS and MGC803 gastric cancer cell lines and the GES-1 gastric mucosa cell line were selected for study. At first, CAC1 expressions of those cell lines were examined by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and western blot examinations, then CAC1 small interfering RNA (CAC1-siRNA) were designed and transfected into the AGS cell line with a relatively high level of CAC1. Once CAC1 was silenced, a series of biological characteristics of AGS cells such as cell proliferation, cell cycle, apoptosis, and expressions of apoptosis-related genes (P53, BCL2 and BAX) were determined by MTT, flow cytometry, qRT-PCR and western blot, respectively.

Results

CAC1 expression of AGS or MGC803 was much higher than that of GES-1. After CAC1 expression was effectively depressed by RNA interference in AGS cells, significant cell growth inhibition occurred. Furthermore, the proportion of cells treated with CAC1-siRNA increased in the G1 phase and decreased in the S phase, indicative of G1 cell cycle arrest. More importantly, the proportions of early/late apoptosis in AGS cells were enhanced with cis-diaminedichloroplatinum (cisplatin, CDDP) treatment, but to a higher extent with cisplatin plus CAC1-siRNA. Interestingly, BCL2 mRNA copies showed about a 30% decrease in the cisplatin group, but dropped by around 60% in the cisplatin plus CAC1-siRNA group. Conversely, the P53 mRNA expressions obtained nearly a two-fold increase in the cisplatin group, in addition to a five-fold increase in the cisplatin plus CAC1-siRNA group, and the BAX mRNA levels had almost a two- and four-fold augmentation, respectively. Meanwhile, P53, BAX and BCL2 showed the same alteration patterns in western blot examinations.

Conclusions

CAC1 can promote cell proliferation in the AGS gastric cancer cell line. Moreover, it can prevent AGS cells from experiencing cisplatin-induced apoptosis via modulating expressions of P53, BCL2 and BAX.

Background

In a previous in vitro study, we confirmed that small-caliber nanofibrous polyurethane (PU) vascular grafts have favorable mechanical properties and biocompatibility. In the present study, we examined the in vivo biocompatibility and stability of these grafts.

Methods

Forty-eight adult male beagle dogs were randomly divided into two groups receiving, respectively, polyurethane (PU) or polytetrafluoroethylene (PTFE) grafts (n = 24 animals / group). Each group was studied at 4, 8, 12, and 24 weeks after graft implantation. Blood flow was analyzed by color Doppler ultrasound and computed tomography angiography. Patency rates were judged by animal survival rates. Coverage with endothelial and smooth muscle cells was characterized by hematoxylin-eosin and immunohistological staining, and scanning electron microscopy (SEM).

Results

Patency rates were significantly higher in the PU group (p = 0.02 vs. PTFE group). During the first 8 weeks, endothelial cells gradually formed a continuous layer on the internal surface of PU grafts, whereas coverage of PTFE graft by endothelial cells was inhomogeneous. After 12 weeks, neointimal thickness remained constant in the PU group, while PTFE group showed neointimal hyperplasia. At 24 weeks, some anastomotic sites of PTFE grafts became stenotic (p = 0.013 vs. PU group). Immunohistological staining revealed a continuous coverage by endothelial cells and an orderly arrangement of smooth muscle cells on PU grafts. Further, SEM showed smooth internal surfaces in PU grafts without thrombus or obvious neointimal hyperplasia.

Conclusions

Small-caliber nanofibrous PU vascular grafts facilitate the endothelialization process, prevent excessive neointimal hyperplasia, and improve patency rates.

Purpose

Sipuleucel-T, the first FDA-approved autologous cellular immunotherapy for treatment of advanced prostate cancer, is manufactured by activating peripheral blood mononuclear cells, including antigen presenting cells (APCs), with a fusion protein containing prostatic acid phosphatase. Analysis of data from three phase 3 trials was performed to immunologically characterize this therapy during the course of the three doses, and to relate the immunological responses to overall survival (OS).

Methods

Sipuleucel-T product characteristics [APC numbers, APC activation (CD54 upregulation), and total nucleated cell (TNC) numbers] were assessed in three randomized, controlled phase 3 studies (N = 737). Antigen-specific cellular and humoral responses were assessed in a subset of subjects. The relationships between these parameters and OS were assessed.

Results

APC activation occurred in the first dose preparation [6.2-fold, (4.65, 7.70); median (25th, 75th percentile)] and increased in the second [10.6-fold (7.83, 13.65)] and third [10.5-fold (7.89, 13.65)] dose preparations. Cytokines and chemokines associated with activated APCs were produced during the manufacture of each dose; T-cell activation-associated cytokines were detected in the second and third dose preparations. Antigen-specific T cells were detectable after administration of the first sipuleucel-T dose. Cumulative APC activation, APC number, and TNC number correlated with OS (P < 0.05). Antigen-specific immune responses were observed in 78.8 % of monitored subjects and their presence correlated with OS (P = 0.003).

Conclusion

Sipuleucel-T broadly engages the immune system by activating APCs ex vivo and inducing long-lived immune responses in vivo. These data indicate antigen-specific immune activation as a mechanism by which sipuleucel-T prolongs OS.

Electronic supplementary material

The online version of this article (doi:10.1007/s00262-012-1317-2) contains supplementary material, which is available to authorized users.

In the host immune system, the leukocytes are often exposed to multiple pathogens including bacteria and viruses. The principal challenge for the host is to efficiently detect the invading pathogen and mount a rapid defensive response. Leukocytes recognize invading pathogens by directly interacting with pathogen-associated molecular patterns via Toll-like receptors (TLRs) expressed on the leukocyte surfaces. In this study, we provide direct evidence that bacterial LPS enhances the host antiviral response by up-regulating TLR3 expression in human peripheral blood monocytes and monocytic cell lines, THP1 cells. Moreover, LPS induces TLR3 expression via a TLR4-MyD88-IRAK-TRAF6-NF-κB-dependent signaling pathway. Interestingly, CYLD, an important deubiquitinase, acts as a negative regulator of TLR3 induction by LPS. Our study thus provides new insights into a novel role for bacterial infection in enhancing host antiviral response; furthermore, it identifies CYLD for the first time as a critical negative regulator of bacterial LPS-induced response.

Sixteen children (17 age mates, 17 vocabulary mates) with specific language impairment (SLI) participated in two studies. In the first, they named fantasy objects. All groups coined novel noun–noun compounds on a majority of trials but only the SLI group had difficulty ordering the nouns as dictated by semantic context. In the second study, the children described the meaning of conventional noun–noun compounds. The SLI and AM groups did not differ in parsing the nouns, but the SLI group was poorer at explaining the semantic relationships between them. Compared to vocabulary mates, a larger proportion of the SLI group successfully parsed the compounds but a smaller proportion could explain them. These difficulties may reflect problems in the development of links within the semantic lexicon.

In situ biomonitoring has been used to assess the effects of pollution on aquatic species in heavily polluted waterways. In the current study, we used in situ biomonitoring in conjunction with molecular biomarker analysis to determine the effects of pollutant exposure in salmon caged in the Duwamish waterway, a Pacific Northwest Superfund site that has been subject to remediation. The Duwamish waterway is an important migratory route for Pacific salmon and has received historic inputs of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs). Juvenile pre-smolt Chinook salmon (Oncorhynchus tshawytscha) caged for 8 days in the three contaminated sites in close proximity within the Duwamish were analyzed for steady state hepatic mRNA expression of 7 exposure biomarker genes encompassing several gene families and known to be responsive to pollutants, including cytochrome P4501A (CYP1A) and CYP2K1, glutathione S-transferase π class (GST pi), microsomal GST (mGST), glutamylcysteine ligase catalytic subunit (GCLC), UDP-glucuronyltransferase family 1 (UDPGT), and type 2 deiodinase (type 2 DI, or D2). Quantitation of gene expression was accomplished by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) in assays developed specifically for Chinook salmon genes. Gill PAH-DNA adducts were assessed as a chemical effects biomarker using 32P-postlabeling. The biomarkers in the field-caged fish were analyzed with respect to caged animals maintained at the hatchery receiving flow-through water. Chemical analysis of sediment samples from three field sampling sites revealed relatively high concentrations of total PAHs in one site (site B2, 6711 ng/g dry weight) and somewhat lower concentrations of PAHs in two adjacent sites (sites B3 and B4, 1482 and 1987 ng/g, respectively). In contrast, waterborne PAHs at all of the sampling sites were relatively low (<1 ng/L). Sediment PCBs at the sites ranged from a low of 421 ng/g at site B3, to 1160 ng/g at site B4, and there were no detectable waterborne PCBs at any of the sites (detection limit=10 ng/L). There were no significant differences (P<0.05) in biomarker gene expression in the Duwamish-caged fish relative to controls, although there was a pattern of gene expression suppression at site B3, the most heavily PAH-enriched site. The lack of a marked perturbation of mRNA biomarkers was consistent with relatively low levels of gill PAH-DNA adduct levels that did not differ among caged reference and field fish, and which were also consistent with relatively low waterborne concentrations of chemicals. The results of our study suggest a low bioavailability of sediment pollutants in caged juvenile Chinook potentially reflecting low waterborne exposures occurring at contaminated sites within the Duwamish waterway that have undergone partial remediation.

Background. To examine the results of treatment of Extramammary Paget's disease (EMPD) in ethnic Chinese. Method. Between 1990 and 2010, patients treated for EMPD were reviewed. Data were analyzed retrospectively. Results. Forty-eight patients were treated by surgical resection. Local recurrence rate was 14.6%. The postresection defects were repaired by primary closure (8.3%), partial thickness skin graft (72.9%), or local/regional flaps (18.8%). Dermal invasion was found in 9 patients (18.8%). Seven patients (14.6%) developed regional lymph node metastasis (concurrent with surgery, n = 1; subsequent to surgery, n = 6), and 3 patients (6.3%) had systemic metastasis after surgery. The presence of dermal invasion was associated with significantly higher incidence of regional lymph nodes and systemic metastasis. The incidence of associated internal malignancy was 8.3%. Conclusion. The mainstay of treatment for EMPD is surgery. Pathological dermal invasion increases the chance of regional lymph node as well as systemic metastasis. The association with internal malignancy warrants preoperative endoscopic examination in all patients.

Originally developed to regulate neuronal excitability, optogenetics is increasingly also used to control other cellular processes with unprecedented spatiotemporal resolution. Optogenetic modulation of all major G-protein signalling pathways (Gq, Gi and Gs) has been achieved using variants of mammalian rod opsin. We show here that the light response driven by such rod opsin-based tools dissipates under repeated exposure, consistent with the known bleaching characteristics of this photopigment. We continue to show that replacing rod opsin with a bleach resistant opsin from Carybdea rastonii, the box jellyfish, (JellyOp) overcomes this limitation. Visible light induced high amplitude, reversible, and reproducible increases in cAMP in mammalian cells expressing JellyOp. While single flashes produced a brief cAMP spike, repeated stimulation could sustain elevated levels for 10s of minutes. JellyOp was more photosensitive than currently available optogenetic tools, responding to white light at irradiances ≥1 µW/cm2. We conclude that JellyOp is a promising new tool for mimicking the activity of Gs-coupled G protein coupled receptors with fine spatiotemporal resolution.

Female gametophyte is the multicellular haploid structure that can produce embryo and endosperm after fertilization, which has become an attractive model system for investigating molecular mechanisms in nuclei migration, cell specification, cell-to-cell communication and many other processes. Previous reports found that the small ubiquitin-like modifier (SUMO) E3 ligase, SIZ1, participated in many processes depending on particular target substrates and suppression of salicylic acid (SA) accumulation. Here, we report that SIZ1 mediates the reproductive process. SIZ1 showed enhanced expression in female organs, but was not detected in the anther or pollen. A defect in the siz1-2 maternal source resulted in reduced seed-set regardless of high SA concentration within the plant. Moreover, aniline blue staining and scanning electron microscopy revealed that funicular and micropylar pollen tube guidance was arrested in siz1-2 plants. Some of the embryo sacs of ovules in siz1-2 were also disrupted quickly after stage FG7. There was no significant affects of the siz1-2 mutation on expression of genes involved in female gametophyte development- or pollen tube guidance in ovaries. Together, our results suggest that SIZ1 sustains the stability and normal function of the mature female gametophyte which is necessary for pollen tube guidance.

It has been a common practice among the oncologist to reduce the dosage of adjuvant radiotherapy for patients after free jejunal flap reconstruction. The current aims to study potential risk of radiation to the visceral flap and the subsequent oncological outcome. Between 1996 and 2010, consecutive patients with carcinoma of the hypopharynx requiring laryngectomy, circumferential pharyngectomy and post-operative irradiation were recruited. Ninety-six patients were recruited. TNM tumor staging at presentation was: stage II (40.6%), stage III (34.4%) and stage IV (25.0%). Median follow-up period after surgery was 68 months. After tumor ablation, reconstruction was performed using free jejunal flap (60.4%), pectoralis major myocutaneous (PM) flap (31.3%) and free anterolateral thigh (ALT) flap (8.3%). All patients underwent adjuvant radiotherapy within 6.4 weeks after surgery. The mean total dose of radiation given to those receiving cutaneous and jejunal flap reconstruction was 62.2 Gy and 54.8 Gy, respectively. There was no secondary ischaemia or necrosis of the flaps after radiotherapy. The 5-year actuarial loco-regional tumor control for the cutaneous flap and jejunal flap group was: stage II (61 vs. 69%, p = 0.9), stage III (36 vs. 46%, p = 0.2) and stage IV (32 vs. 14%, p = 0.04), respectively. Reduction of radiation dosage in free jejunal group adversely affects the oncological control in stage IV hypopharyngeal carcinoma. In such circumstances, tubed cutaneous flaps are the preferred reconstructive option, so that full-dose radiotherapy can be given.

Introduction

Tobacco smoke is known to be the main cause of lung, head and neck tumors. Recently, evidence for an increasing breast cancer risk associated with tobacco smoke exposure has been emerging. We and other groups have shown that nicotine, as a non-conventional carcinogen, has the potential to facilitate cancer genesis and progression. However, the underlying mechanisms by which the smoke affects the breast, rather than the lung, remain unclear. Here, we examine possible downstream signaling pathways of the nicotinic acetylcholine receptor (nAChR) and their role in breast cancer promotion.

Methods

Using human benign MCF10A and malignant MDA-MB-231 breast cells and specific inhibitors of possible downstream kinases, we identified nAChR effectors that were activated by treatment with nicotine. We further tested the effects of these effector pathways on the regulation of E2F1 activation, cell cycle progression and on Bcl-2 expression and long-term cell survival.

Results

In this study, we demonstrated a novel signaling mechanism by which nicotine exposure activated Src to sensitize epidermal growth factor receptor (EGFR)-mediated pathways for breast cancer cell growth promotion. After the ligation of nAChR with nicotine, EGFR was shown to be activated and then internalized in both MCF10A and MDA-MB-231 breast cancer cells. Subsequently, Src, Akt and ERK1/2 were phosphorylated at different time points following nicotine treatment. We further demonstrated that through Src, the ligation of nicotine with nAChR stimulated the EGFR/ERK1/2 pathway for the activation of E2F1 and further cell progression. Our data also showed that Akt functioned directly downstream of Src and was responsible for the increase of Bcl-2 expression and long-term cell survival.

Conclusions

Our study reveals the existence of a potential, regulatory network governed by the interaction of nicotine and nAChR that integrates the conventional, mitogenic Src and EGFR signals for breast cancer development.

Objective

Evaluation of single nucleotide polymorphisms (SNPs) in the interleukin-10 promoter (−592 and −819) on risk for death after burn injury.

Methods

Association between the IL-10 SNPs and outcome after burn injury was evaluated in a cohort of 265 patients from Parkland Hospital, Dallas, TX with ≥15% TBSA burns without non-burn trauma (ISS ≤ 16), traumatic or anoxic brain injury or spinal cord injury, who survived >48 h under an IRB-approved protocol. Clinical data were collected prospectively and genotyping was conducted by TaqMan assay. Whole blood from 31 healthy volunteers was stimulated with LPS (100 ng/mL) to determine the level of IL-10 expression for each allele by enzyme-linked immunosorbent assay (ELISA).

Results

After adjustment for percent total body surface area (TBSA) burned, inhalation injury, age, gender, and race/ethnicity, carriage of −592A and/or −819T was significantly associated (P = 0.014) with a decreased risk for death (adjusted odds ratio: 0.404; 95% CI: 0.197–0.829). As the candidate SNPs were in complete linkage disequilibrium, it was not possible to distinguish which allele was associated with decreased mortality risk. Age, inhalation injury, and full-thickness burn size were significantly associated with increased risk for death. In the LPS stimulated blood of healthy controls, carriage of the −592A and/or −819T allele demonstrated a trend for decreased levels of IL-10 (P = 0.079).

Conclusion

Carriage of the −592A and/or −819T allele in the IL-10 promoter appears to reduce the risk for death after burn injury.

Objectives. This study aims to report our experience in the management of HNBCC in ethnic Chinese over a 10-year period. Methods. A retrospective review of all ethnic Chinese patients with HNBCC treated in a tertiary centre from 1999 to 2009. Results. From 1999 to 2009, 225 patients underwent surgical excision for HNBCC. Majority were elderly female patients. Commonest presentation was a pigmented (76.2%) ulcer (64.8%) over the nose (31.6%). Median skin margin taken on tumour excision was 2.0 mm; primary skin closure was achieved in 51.8%. Postresection skin margin was clear in 75.4%. Of those with inadequate skin margins, 56.7% opted for further treatment, 43.4% for observation. Recurrence rates were 2.6% and 13.8%, respectively (P = 0.106). Overall recurrence rate was 5.5%. Conclusions. HNBCC commonly presented as pigmented ulcers over the nose of elderly female patients in our locality. Adequate tumour excision ± reconstruction offered the best chance of cure. Reexcision of those with inadequate skin margins improved local tumour control.

Stimulation of phagocytic leukocytes with bacterial chemoattractant resulted in the release of matrix metal-loproteinases (MMPs). Little is known about the mechanisms of bacterial chemoattractant regulation of MMP in phagocytic leukocytes. We report here that the mechanisms of the bacterial chemotactic peptidefMLP-induced MMP -9 release in monocytes appeared to be different from fMLP-stimulated MMP-9 release in neutrophils. In freshly prepared peripheral blood monocytes, fMLP induces MMP-9 release, starting at 8 h after stimulation. These functions of fMLP is accompanied by an increase in TNFα expression, and mediated through the phosphorylation of ERK1/2 in monocytes. However, neutrophil preparations that responded to fMLP with MMP-9 release did not require activation of ERK1/2 and TNFα expression. These results suggest a different role of fMLP in MMP-9 expression in neutrophils and monocytes, and the signal molecules involved in mediating this effect in human blood monocytes stimulated by bacterial chemoattractant.

We present an applied study in cancer genomics for integrating data and inferences from laboratory experiments on cancer cell lines with observational data obtained from human breast cancer studies. The biological focus is on improving understanding of transcriptional responses of tumors to changes in the pH level of the cellular microenvironment. The statistical focus is on connecting experimentally defined biomarkers of such responses to clinical outcome in observational studies of breast cancer patients. Our analysis exemplifies a general strategy for accomplishing this kind of integration across contexts. The statistical methodologies employed here draw heavily on Bayesian sparse factor models for identifying, modularizing and correlating with clinical outcome these signatures of aggregate changes in gene expression. By projecting patterns of biological response linked to specific experimental interventions into observational studies where such responses may be evidenced via variation in gene expression across samples, we are able to define biomarkers of clinically relevant physiological states and outcomes that are rooted in the biology of the original experiment. Through this approach we identify microenvironment-related prognostic factors capable of predicting long term survival in two independent breast cancer datasets. These results suggest possible directions for future laboratory studies, as well as indicate the potential for therapeutic advances though targeted disruption of specific pathway components.

Although lactic acidosis is a prominent feature of solid tumors, we still have limited understanding of the mechanisms by which lactic acidosis influences metabolic phenotypes of cancer cells. We compared global transcriptional responses of breast cancer cells in response to three distinct tumor microenvironmental stresses: lactic acidosis, glucose deprivation, and hypoxia. We found that lactic acidosis and glucose deprivation trigger highly similar transcriptional responses, each inducing features of starvation response. In contrast to their comparable effects on gene expression, lactic acidosis and glucose deprivation have opposing effects on glucose uptake. This divergence of metabolic responses in the context of highly similar transcriptional responses allows the identification of a small subset of genes that are regulated in opposite directions by these two conditions. Among these selected genes, TXNIP and its paralogue ARRDC4 are both induced under lactic acidosis and repressed with glucose deprivation. This induction of TXNIP under lactic acidosis is caused by the activation of the glucose-sensing helix-loop-helix transcriptional complex MondoA:Mlx, which is usually triggered upon glucose exposure. Therefore, the upregulation of TXNIP significantly contributes to inhibition of tumor glycolytic phenotypes under lactic acidosis. Expression levels of TXNIP and ARRDC4 in human cancers are also highly correlated with predicted lactic acidosis pathway activities and associated with favorable clinical outcomes. Lactic acidosis triggers features of starvation response while activating the glucose-sensing MondoA-TXNIP pathways and contributing to the “anti-Warburg” metabolic effects and anti-tumor properties of cancer cells. These results stem from integrative analysis of transcriptome and metabolic response data under various tumor microenvironmental stresses and open new paths to explore how these stresses influence phenotypic and metabolic adaptations in human cancers.

Author Summary

Solid tumors usually have many differences in their chemical environments, such as low oxygen, depletion of glucose, high acidity (low pH), and accumulation of lactate, from normal tissues. These changes are usually called tumor microenvironmental stresses. In this study, we have used microarrays to compare the transcriptional response and metabolic adaptation in response to these different stresses seen in the tumor microenvironments. Through these comparisons, we have found that lactic acidosis triggers a starvation response, highly similar to glucose deprivation, even in the presence of abundant nutrients and oxygen. Even the cells seem to be starved; cells under lactic acidosis have decreased glucose uptake. We found this unexpected biological behavior was due to the paradoxical induction of a glucose-sensing Mondo-TXNIP pathway. The activation of this novel anti-tumor pathway under lactic acidosis contributes to the anti-Warburg effect and the restriction of cell growth in tumorigenesis by limiting nutrient availability and its inactivation may be required for tumor progression under these microenvironmental stresses.

There is overwhelming evidence that tyrosine kinases play an important role in cancer development. As a prototype of targeted therapy, tyrosine kinase inhibitors are now successfully applied to cancer treatment. However, as single agents, tyrosine kinase inhibitors have not achieved satisfactory results in the treatment of prostate cancer, principally due to their inability to efficiently kill tumor cells. The authors’ laboratory has been interested in the role of the Src complex in prostate cancer progression, including the induction of androgen independence and metastasis. Previously, the authors reported that Src inhibitors such as saracatinib and PP2 caused G1 growth arrest and diminished invasiveness in prostate cancer cells but rarely apoptosis. Here, they have shown that Src family kinase (SFK) inhibitors can induce a high level of autophagy, which protects treated cells from undergoing apoptosis. Src siRNA knockdown experiments confirmed that autophagy was indeed caused by the lack of Src activity. The SFK inhibitor-induced autophagy is accompanied by the inhibition of the PI3K (type I)/Akt/mTOR signaling pathway. To test whether autophagy blockade could lead to enhanced cell death, pharmacological inhibitors (3-methyladenine and chloroquine) and a genetic inhibitor (siRNA targeting Atg7) were used in combination with SFK inhibitors. The results showed that autophagy inhibition effectively enhanced cell killing induced by SFK inhibitors. Importantly, the authors showed that a combination of saracatinib with chloroquine in mice significantly reduced prostate cancer (PC3) xenograft growth compared with the control group. Taken together, these data suggest that (1) autophagy serves a protective role in SFK inhibitor-mediated cell killing, and (2) clinically acceptable autophagy modulators may be used beneficially as adjunctive therapeutic agents for SFK inhibitors.

Background

Mitochondria are dynamic organelles that move along actin filaments, and serve as calcium stores in plant cells. The positioning and dynamics of mitochondria depend on membrane-cytoskeleton interactions, but it is not clear whether microfilament cytoskeleton has a direct effect on mitochondrial function and Ca2+ storage. Therefore, we designed a series of experiments to clarify the effects of actin filaments on mitochondrial Ca2+ storage, cytoplasmic Ca2+ concentration ([Ca2+]c), and the interaction between mitochondrial Ca2+ and cytoplasmic Ca2+ in Arabidopsis root hairs.

Results

In this study, we found that treatments with latrunculin B (Lat-B) and jasplakinolide (Jas), which depolymerize and polymerize actin filaments respectively, decreased membrane potential and Ca2+ stores in the mitochondria of Arabidopsis root hairs. Simultaneously, these treatments induced an instantaneous increase of cytoplasmic Ca2+, followed by a continuous decrease. All of these effects were inhibited by pretreatment with cyclosporin A (Cs A), a representative blocker of the mitochondrial permeability transition pore (mPTP). Moreover, we found there was a Ca2+ concentration gradient in mitochondria from the tip to the base of the root hair, and this gradient could be disrupted by actin-acting drugs.

Conclusions

Based on these results, we concluded that the disruption of actin filaments caused by Lat-B or Jas promoted irreversible opening of the mPTP, resulting in mitochondrial Ca2+ release into the cytoplasm, and consequent changes in [Ca2+]c. We suggest that normal polymerization and depolymerization of actin filaments are essential for mitochondrial Ca2+ storage in root hairs.

Background

The Taiwanese government began reimbursement for smoking cessation in 2002. Certification from a training program was required for physicians who wanted reimbursement. The program certified 6,009 physicians till 2007. The objective of this study is to evaluate the short- and long term efficacy of the training program.

Methods

For short term evaluation, all trainees in 2007 were recruited. For long term evaluation, computer randomly selected 2,000 trainees who received training from 2002 to 2006 were recruited. Course satisfaction, knowledge, confidence in providing smoking cessation services and the adherence to a practice guideline were evaluated by questionnaires.

Results

Trainees reported high satisfaction with the training program. There was significant difference between pre- and post-test scores in knowledge. Confidence in providing services was lower in the long term evaluation compared to short term evaluation. For adherence to a practice guideline, 86% asked the status of smoking, 88% advised the smokers to quit, 76% assessed the smoker's willingness to quit, 59% assisted the smokers to quit, and 60% arranged follow-up visits for smokers. The incentive of reimbursement was the most significant factor affecting confidence and adherence.

Conclusions

The training program was satisfactory and effective. Adherence to a practice guideline in our study was better than studies without physician training in other countries.

There is overwhelming evidence that tyrosine kinases play an important role in cancer development. As a prototype of targeted therapy, tyrosine kinase inhibitors are now successfully applied to cancer treatment. However, as single agents, tyrosine kinase inhibitors have not achieved satisfactory results in the treatment of prostate cancer, principally due to their inability to efficiently kill tumor cells. The authors’ laboratory has been interested in the role of the Src complex in prostate cancer progression, including the induction of androgen independence and metastasis. Previously, the authors reported that Src inhibitors such as saracatinib and PP2 caused G1 growth arrest and diminished invasiveness in prostate cancer cells but rarely apoptosis. Here, they have shown that Src family kinase (SFK) inhibitors can induce a high level of autophagy, which protects treated cells from undergoing apoptosis. Src siRNA knockdown experiments confirmed that autophagy was indeed caused by the lack of Src activity. The SFK inhibitor-induced autophagy is accompanied by the inhibition of the PI3K (type I)/Akt/mTOR signaling pathway. To test whether autophagy blockade could lead to enhanced cell death, pharmacological inhibitors (3-methyladenine and chloroquine) and a genetic inhibitor (siRNA targeting Atg7) were used in combination with SFK inhibitors. The results showed that autophagy inhibition effectively enhanced cell killing induced by SFK inhibitors. Importantly, the authors showed that a combination of saracatinib with chloroquine in mice significantly reduced prostate cancer (PC3) xenograft growth compared with the control group. Taken together, these data suggest that (1) autophagy serves a protective role in SFK inhibitor-mediated cell killing, and (2) clinically acceptable autophagy modulators may be used beneficially as adjunctive therapeutic agents for SFK inhibitors.

Schistosoma japonicum is the most pathogenic agent of hepatosplenic schistosomiasis that has killed millions of people in China. It causes extended fibrosis in the periportal space (PPF) and in the hepatic parenchyma (ParF). The deposition of the extracellular matrix proteins composing the fibrotic scar is regulated by cytokines and chemokines, the production of which may play a key role in disease progression. We investigated whether fibrosis and splenomegaly in fishermen living in an endemic region for S. japonicum were associated with abnormal cytokine/chemokine production by blood mononuclear cells. PPF and ParF were considered as separate fibrosis phenotypes. PPF was associated (by univariate analysis) with low levels of IL-10 (p=4 × 10-4), RANTES (p=4 × 10-4) and MIP-1α (p=7 × 10-3) production in cultures of blood leukocytes stimulated with schistosome egg antigens. Logistic regression that included exposure, anti-schistosome treatments and drinking habits as covariates showed that IL-10 exhibited the strongest association with PPF (p=1 × 10-4, OR=10.8, CI=3.2-38). Splenomegaly was associated with low levels of IL-10 production (p=4 × 10-3) even in the presence of PPF as covariate (p=0.01, OR=3.5, 1.3-8.9), indicating a probable direct relationship between IL-10 and splenomegaly. Furthermore, ParF was associated with low levels of production for IFN-γ (p=3.5 10-3; OR= 8.2; 2-33) but not for IL-10 or RANTES. These data are consistent with IL-10 playing a key role in the development of severe hepatic and spleen disease and differences in the cytokine-mediated control of PPF and ParF in humans infected with S. japonicum.

Human disease studies using DNA microarrays in both clinical/observational and experimental/controlled studies are having increasing impact on our understanding of the complexity of human diseases. A fundamental concept is the use of gene expression as a “common currency” that links the results of in vitro controlled experiments to in vivo observational human studies. Many studies – in cancer and other diseases – have shown promise in using in vitro cell manipulations to improve understanding of in vivo biology, but experiments often simply fail to reflect the enormous phenotypic variation seen in human diseases. We address this with a framework and methods to dissect, enhance and extend the in vivo utility of in vitro derived gene expression signatures. From an experimentally defined gene expression signature we use statistical factor analysis to generate multiple quantitative factors in human cancer gene expression data. These factors retain their relationship to the original, one-dimensional in vitro signature but better describe the diversity of in vivo biology. In a breast cancer analysis, we show that factors can reflect fundamentally different biological processes linked to molecular and clinical features of human cancers, and that in combination they can improve prediction of clinical outcomes.