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The low bioavailability of most phytochemicals limits their anticancer effects in humans. The present study was designed to test whether combining arctigenin (Arc), a lignan mainly from the seed of Arctium lappa, with green tea (GT) and quercetin (Q) enhances the chemopreventive effect on prostate cancer. We performed in vitro proliferation studies on different cell lines. We observed a strong synergistic anti-proliferative effect of GT+Q+Arc in exposing androgen-sensitive human prostate cancer LNCaP cells. The pre-malignant WPE1-NA22 cell line was more sensitive to this combination. No cytotoxicity was observed in normal prostate epithelial PrEC cells. For an in vivo study, 3-week-old, prostate-specific PTEN (phosphatase and tensin homolog) knockout mice were treated with GT+Q, Arc, GT+Q+Arc, or the control daily until 16 weeks of age. In vivo imaging using prostate-specific membrane antigen (PSMA) probes demonstrated that the prostate tumorigenesis was significantly inhibited by 40% (GT+Q), 60% (Arc at 30 mg/kg bw), and 90% (GT+Q+Arc) compared to the control. A pathological examination showed that all control mice developed invasive prostate adenocarcinoma. In contrast, the primary lesion in the GT+Q and Arc alone groups was high-grade prostatic intraepithelial neoplasia (PIN), with low-grade PIN in the GT+Q+Arc group. The combined effect of GT+Q+Arc was associated with an increased inhibition of the androgen receptor, the PI3K/Akt pathway, Ki67 expression, and angiogenesis. This study demonstrates that combining Arc with GT and Q was highly effective in prostate cancer chemoprevention. These results warrant clinical trials to confirm the efficacy of this combination in humans.

Toxin cargo genes are often horizontally transferred by phages between bacterial species and are known to play an important role in the evolution of bacterial pathogenesis. Here, we show how these same genes have been horizontally transferred from phage or bacteria to animals and have resulted in novel adaptations. We discovered that two widespread bacterial genes encoding toxins of animal cells, cytolethal distending toxin subunit B (cdtB) and apoptosis-inducing protein of 56 kDa (aip56), were captured by insect genomes through horizontal gene transfer from bacteria or phages. To study the function of these genes in insects, we focused on Drosophila ananassae as a model. In the D. ananassae subgroup species, cdtB and aip56 are present as singular (cdtB) or fused copies (cdtB::aip56) on the second chromosome. We found that cdtB and aip56 genes and encoded proteins were expressed by immune cells, some proteins were localized to the wasp embryo's serosa, and their expression increased following parasitoid wasp infection. Species of the ananassae subgroup are highly resistant to parasitoid wasps, and we observed that D. ananassae lines carrying null mutations in cdtB and aip56 toxin genes were more susceptible to parasitoids than the wild type. We conclude that toxin cargo genes were captured by these insects millions of years ago and integrated as novel modules into their innate immune system. These modules now represent components of a heretofore undescribed defense response and are important for resistance to parasitoid wasps. Phage or bacterially derived eukaryotic toxin genes serve as macromutations that can spur the instantaneous evolution of novelty in animals.

Background & aimsNonalcoholic steatohepatitis (NASH) is a complex disease involving both genetic and environmental factors in its onset and progression. We analyzed NASH phenotypes in a genetically diverse cohort of mice, the Hybrid Mouse Diversity Panel, to identify genes contributing to disease susceptibility.MethodsA "systems genetics" approach, involving integration of genetic, transcriptomic, and phenotypic data, was used to identify candidate genes and pathways in a mouse model of NASH. The causal role of Matrix Gla Protein (MGP) was validated using heterozygous MGP knockout (Mgp+/-) mice. The mechanistic role of MGP in transforming growth factor-beta (TGF-β) signaling was examined in the LX-2 stellate cell line by using a loss of function approach.ResultsLocal cis-acting regulation of MGP was correlated with fibrosis, suggesting a causal role in NASH, and this was validated using loss of function experiments in 2 models of diet-induced NASH. Using single-cell RNA sequencing, Mgp was found to be primarily expressed in hepatic stellate cells and dendritic cells in mice. Knockdown of MGP expression in stellate LX-2 cells led to a blunted response to TGF-β stimulation. This was associated with reduced regulatory SMAD phosphorylation and TGF-β receptor ALK1 expression as well as increased expression of inhibitory SMAD6. Hepatic MGP expression was found to be significantly correlated with the severity of fibrosis in livers of patients with NASH, suggesting relevance to human disease.ConclusionsMGP regulates liver fibrosis and TGF-β signaling in hepatic stellate cells and contributes to NASH pathogenesis.

AimsNon-alcoholic steatohepatitis (NASH) is associated with increased overall morbidity and mortality in non-alcoholic fatty liver disease (NAFLD) patients. Liver fibrosis is the strongest prognostic factor for clinical outcomes, liver-related mortality and liver transplantation. Currently, no single therapy or medication for NASH has been approved by the U.S. Food and Drug Administration (FDA). Oxy210, an oxysterol derivative, displays the unique property of antagonizing both Hedgehog (Hh) and transforming growth factor-beta (TGF-β) signalling in primary human hepatic stellate cells (HSC). We hypothesized that inhibition of both Hh and TGF-β signalling by Oxy210 could reduce hepatic fibrosis in NASH. In this study, we examined the therapeutic potential of Oxy210 on NASH in vivo.MethodsWe examined the effect of Oxy210 treatment on Hh and TGF-β pathways in HSC. The efficacy of Oxy210 on liver fibrosis was tested in a 'humanized' hyperlipidemic mouse model of NASH that has high relevance to human pathology.Approach and resultsWe show that Oxy210 inhibits both Hh and TGF-β pathways in human HSC and attenuates baseline and TGF-β-induced expression of pro-fibrotic genes in vitro. Oral delivery of Oxy210 in food resulted in significant liver exposure and significantly reduced hepatic fibrosis in mice over the course of the 16-week study with no apparent safety issues. Additionally, we observed several benefits related to NASH phenotype: (a) reduced plasma pro-inflammatory cytokine and the corresponding hepatic gene expression; (b) reduced pro-fibrotic cytokine and inflammasome gene expression in the liver; (c) reduced apoptosis in the liver; (d) reduced hepatic unesterified cholesterol accumulation; and (e) reduced plasma total and unesterified cholesterol levels.ConclusionsOxy210 effectively ameliorated hepatic fibrosis and inflammation and improved hypercholesterolemia in mice. Our findings suggest that Oxy210 and related analogues are a new class of drug candidates that may serve as potential therapeutics candidates for NASH.

Background & aimsLiver fibrosis is a multifactorial trait that develops in response to chronic liver injury. Our aim was to characterize the genetic architecture of carbon tetrachloride (CCl4)-induced liver fibrosis using the Hybrid Mouse Diversity Panel, a panel of more than 100 genetically distinct mouse strains optimized for genome-wide association studies and systems genetics.MethodsChronic liver injury was induced by CCl4 injections twice weekly for 6 weeks. Four hundred thirty-seven mice received CCl4 and 256 received vehicle, after which animals were euthanized for liver histology and gene expression. Using automated digital image analysis, we quantified fibrosis as the collagen proportionate area of the whole section, excluding normal collagen.ResultsWe discovered broad variation in fibrosis among the Hybrid Mouse Diversity Panel strains, demonstrating a significant genetic influence. Genome-wide association analyses revealed significant and suggestive loci underlying susceptibility to fibrosis, some of which overlapped with loci identified in mouse crosses and human population studies. Liver global gene expression was assessed by RNA sequencing across the strains, and candidate genes were identified using differential expression and expression quantitative trait locus analyses. Gene set enrichment analyses identified the underlying pathways, of which stellate cell involvement was prominent, and coexpression network modeling identified modules associated with fibrosis.ConclusionsOur results provide a rich resource for the design of experiments to understand mechanisms underlying fibrosis and for rational strain selection when testing antifibrotic drugs.

Patients with chronic kidney disease (CKD) have elevated circulating levels of trimethylamine N-oxide (TMAO), a metabolite derived from gut microbes and associated with cardiovascular diseases. High circulating levels of TMAO and its dietary precursor, choline, predict increased risk for development of CKD in apparently healthy subjects, and studies in mice fed TMAO or choline suggest that TMAO can contribute to kidney impairment and renal fibrosis. Here we examined the interactions between TMAO, kidney disease, and cardiovascular disease in mouse models. We observed that while female hyperlipidemic apoE KO mice fed a 0.2% adenine diet for 14 weeks developed CKD with elevated plasma levels of TMAO, provision of a non-lethal inhibitor of gut microbial trimethylamine (TMA) production, iodomethylcholine (IMC), significantly reduced multiple markers of renal injury (plasma creatinine, cystatin C, FGF23, and TMAO), reduced histopathologic evidence of fibrosis, and markedly attenuated development of microalbuminuria. In addition, while the adenine-induced CKD model significantly increased heart weight, a surrogate marker for myocardial hypertrophy, this was largely prevented by IMC supplementation. Surprisingly, adenine feeding did not increase atherosclerosis and significantly decreased the expression of inflammatory genes in the aorta compared to the control groups, effects unrelated to TMAO levels. Our data demonstrate that inhibition of TMAO production attenuated CKD development and cardiac hypertrophy in mice, suggesting that TMAO reduction may be a novel strategy in treating CKD and its cardiovascular disease complications.

BackgroundNormal tissue toxicity is an inevitable consequence of primary or secondary brain tumor radiotherapy. Cranial irradiation commonly leads to neurocognitive deficits that manifest months or years after treatment. Mechanistically, radiation-induced loss of neural stem/progenitor cells, neuroinflammation, and demyelination are contributing factors that lead to progressive cognitive decline.MethodsThe effects of 1-[(4-nitrophenyl)sulfonyl]-4-phenylpiperazine (NSPP) on irradiated murine neurospheres, microglia cells, and patient-derived gliomaspheres were assessed by sphere-formation assays, flow cytometry, and interleukin (IL)-6 enzyme-linked immunosorbent assay. Activation of the hedgehog pathway was studied by quantitative reverse transcription PCR. The in vivo effects of NSPP were analyzed using flow cytometry, sphere-formation assays, immunohistochemistry, behavioral testing, and an intracranial mouse model of glioblastoma.ResultsWe report that NSPP mitigates radiation-induced normal tissue toxicity in the brains of mice. NSPP treatment significantly increased the number of neural stem/progenitor cells after brain irradiation in female animals, and inhibited radiation-induced microglia activation and expression of the pro-inflammatory cytokine IL-6. Behavioral testing revealed that treatment with NSPP after radiotherapy was able to successfully mitigate radiation-induced decline in memory function of the brain. In mouse models of glioblastoma, NSPP showed no toxicity and did not interfere with the growth-delaying effects of radiation.ConclusionsWe conclude that NSPP has the potential to mitigate cognitive decline in patients undergoing partial or whole brain irradiation without promoting tumor growth and that the use of this compound as a radiation mitigator of radiation late effects on the central nervous system warrants further investigation.

PurposeProstate-specific membrane antigen (PSMA) targeting radioligands deliver radiation to PSMA-expressing cells. However, the relationship between PSMA levels and intralesion heterogeneity of PSMA expression, and cytotoxic radiation by radioligand therapy (RLT) is unknown. Here we investigate RLT efficacy as function of PSMA levels/cell, and the fraction of PSMA+ cells in a tumor.Experimental designRM1 cells expressing different levels of PSMA (PSMA-, PSMA+, PSMA++, PSMA+++; study 1) or a mix of PSMA+ and PSMA- RM1 (study 2, 4) or PC-3/PC-3-PIP (study 3) cells at various ratios were injected into mice. Mice received 177Lu- (studies 1-3) or 225Ac- (study 4) PSMA617. Tumor growth was monitored. Two days post-RLT, tumors were resected in a subset of mice. Radioligand uptake and DNA damage were quantified.Results177Lu-PSMA617 efficacy increased with increasing PSMA levels (study 1) and fractions of PSMA positive cells (studies 2, 3) in both, the RM1 and PC-3-PIP models. In tumors resected 2 days post-RLT, PSMA expression correlated with 177Lu-PSMA617 uptake and the degree of DNA damage. Compared with 177Lu-PSMA617, 225Ac-PSMA617 improved overall antitumor effectiveness and tended to enhance the differences in therapeutic efficacy between experimental groups.ConclusionsIn the current models, both the degree of PSMA expression and the fraction of PSMA+ cells correlate with 177Lu-/225Ac-PSMA617 tumor uptake and DNA damage, and thus, RLT efficacy. Low or heterogeneous PSMA expression represents a resistance mechanism to RLT.See related commentary by Ravi Kumar and Hofman, p. 2774.

This study investigated the inhibitory effect of arctigenin, a novel anti-inflammatory lignan, on prostate cancer in obese conditions both in vitro and in vivo. In vitro obese models were established by co-culture of mouse adipocytes 3T3-L1 with androgen-sensitive LNCaP human prostate cancer cells, or by culturing LNCaP cells in adipocytes-conditioned medium. Arctigenin significantly inhibited LNCaP proliferation, along with decreased androgen receptor (AR) and increased Nkx3.1 cellular expression. Male severe combined immunodeficiency mice were subcutaneously implanted with human prostate cancer LAPC-4 xenograft tumors for in vivo study. Mice were fed high-fat (HF) diet and orally given arctigenin at 50 mg/kg body weight daily or vehicle control for 6 weeks. Tumor bearing HF control mice showed a significant increase in serum free fatty acids (FFAs) and decrease in subcutaneous/peritoneal fat depots compared to non-tumor bearing control mice. Arctigenin intervention significantly reduced tumor growth by 45%, associated with decreased circulating FFAs and adipokines/cytokines including IGF-1, VEGF, and MCP-1, along with decreased AR, Ki67, and microvessel density and increased Nkx3.1 expression in tumors. These results indicate the strong ability of arctigenin to co-target obesity and tumor itself in inhibition of prostate tumor growth at a lower concentration compared to most phytochemicals.

Background: Evidence suggests the renin-angiotensin system (RAS) plays key immunomodulatory roles. In particular, angiotensin-converting enzyme (ACE) has been shown to play a role in antimicrobial host defense. ACE inhibitors (ACEi) and angiotensin receptor blockers (ARB) are some of the most commonly prescribed medications, especially in patients undergoing invasive surgery. Thus, the current study assessed the immunomodulatory effect of RAS-modulation in a preclinical model of implant infection. Methods: In vitro antimicrobial effects of ACEi and ARBs were first assessed. C57BL/6J mice subsequently received either an ACEi (lisinopril; 16 mg/kg/day), an ARB (losartan; 30 mg/kg/day), or no treatment. Conditioned mice blood was then utilized to quantify respiratory burst function as well as Staphylococcus aureus Xen36 burden ex vivo in each treatment group. S. aureus infectious burden for each treatment group was then assessed in vivo using a validated mouse model of implant infection. Real-time quantitation of infectious burden via bioluminescent imaging over the course of 28 days post-procedure was assessed. Host response via monocyte and neutrophil infiltration within paraspinal and spleen tissue was quantified by immunohistochemistry for F4/80 and myeloperoxidase, respectively. Results: Blood from mice treated with an ACEi demonstrated a decreased ability to eradicate bacteria when mixed with Xen36 as significantly higher levels of colony forming units (CFU) and biofilm formation was appreciated ex vivo (p < 0.05). Mice treated with an ACEi showed a higher infection burden in vivo at all times (p < 0.05) and significantly higher CFUs of bacteria on both implant and paraspinal tissue at the time of sacrifice (p < 0.05 for each comparison). There was also significantly decreased infiltration and respiratory burst function of immune effector cells in the ACEi group (p < 0.05). Conclusion: ACEi, but not ARB, treatment resulted in increased S. aureus burden and impaired immune response in a preclinical model of implant infection. These results suggest that perioperative ACEi use may represent a previously unappreciated risk factor for surgical site infection. Given the relative interchangeability of ACEi and ARB from a cardiovascular standpoint, this risk factor may be modifiable.

BackgroundCancer is frequently associated with tumor-related anemia, and many chemotherapeutic agents impair hematopoiesis, leading to impaired quality of life for affected patients. The use of erythropoiesis-stimulating agents has come under scrutiny after prospective clinical trials using recombinant erythropoietin to correct anemia reported increased incidence of thromboembolic events and cancer-related deaths. Furthermore, previous preclinical reports indicated expansion of the pool of breast cancer-initiating cells when erythropoietin was combined with ionizing radiation.MethodsUsing four established breast cancer cell lines, we test the effects of recombinant human erythropoietin and the number of breast cancer-initiating cells in vitro and in vivo and study if recombinant human erythropoietin promotes the phenotype conversion of non-tumorigenic breast cancer cells into breast cancer-initiating cells. In a prospective study, we evaluate whether elevated endogenous serum erythropoietin levels correlate with increased numbers of tumor-initiating cells in a cohort of breast cancer patients who were scheduled to undergo radiation treatment.ResultsOur results indicate that recombinant erythropoietin increased the number of tumor-initiating cells in established breast cancer lines in vitro. Irradiation of breast cancer xenografts caused a phenotype conversion of non-stem breast cancer cells into induced breast cancer-initiating cells. This effect coincided with re-expression of the pluripotency factors c-Myc, Sox2, and Oct4 and was enhanced by recombinant erythropoietin. Hemoglobin levels were inversely correlated with serum erythropoietin levels, and the latter were correlated with disease stage. However, tumor sections revealed a negative correlation between serum erythropoietin levels and the number of ALDH1A3-positive cells, a marker for breast cancer-initiating cells.ConclusionsWe conclude that physiologically slow-rising serum erythropoietin levels in response to tumor-related or chemotherapy-induced anemia, as opposed to large doses of recombinant erythropoietin, do not increase the pool of breast cancer-initiating cells.

We report the genetic analysis of a "humanized" hyperlipidemic mouse model for progressive nonalcoholic steatohepatitis (NASH) and fibrosis. Mice carrying transgenes for human apolipoprotein E*3-Leiden and cholesteryl ester transfer protein and fed a "Western" diet were studied on the genetic backgrounds of over 100 inbred mouse strains. The mice developed hepatic inflammation and fibrosis that was highly dependent on genetic background, with vast differences in the degree of fibrosis. Histological analysis showed features characteristic of human NASH, including macrovesicular steatosis, hepatocellular ballooning, inflammatory foci, and pericellular collagen deposition. Time course experiments indicated that while hepatic triglyceride levels increased steadily on the diet, hepatic fibrosis occurred at about 12 weeks. We found that the genetic variation predisposing to NASH and fibrosis differs markedly from that predisposing to simple steatosis, consistent with a multistep model in which distinct genetic factors are involved. Moreover, genome-wide association identified distinct genetic loci contributing to steatosis and NASH. Finally, we used hepatic expression data from the mouse panel and from 68 bariatric surgery patients with normal liver, steatosis, or NASH to identify enriched biological pathways. Conclusion: The pathways showed substantial overlap between our mouse model and the human disease.

Parathyroid hormone (PTH) exerts dual effects, anabolic or catabolic, on bone when administrated intermittently or continuously, via mechanisms that remain largely unknown. PTH binding to cells induces PTH-responsive genes including primary response genes (PRGs). PRGs are rapidly induced without the need for de novo protein synthesis, thereby playing pivotal roles in directing subsequent molecular responses. In this study, to understand the role of PRGs in mediating osteoblastic cellular responses to PTH, we investigated whether various durations of PTH differentially induce PRGs in primary osteoblasts and MC3T3-E1. Nurr1 and RANKL, PRGs known for their anabolic and catabolic roles in bone metabolism respectively, presented distinctive transient vs. sustained induction kinetics. Corroborating their roles, maximum induction of Nurr1 was sufficiently achieved by brief PTH in as little as 30 minutes and continued beyond that, while maximum induction of RANKL was achieved only by prolonged PTH over 4 hours. Our data suggested distinctive regulatory mechanisms for Nurr1 and RANKL: PKA-mediated chromatin rearrangement for transcriptional regulation of both PRGs and ERK-mediated transcriptional regulation for RANKL but not Nurr1. Lastly, we classified PRGs into two groups based on the induction kinetics: The group that required brief PTH for maximum induction included Nur77, cox-2, and Nurr1, all of which are reported to play roles in bone formation. The other group that required prolonged PTH for maximum induction included IL-6 and RANKL, which play roles in bone resorption. Together, our data suggested the crucial role of PRG groups in mediating differential osteoblastic cellular responses to intermittent vs. continuous PTH. Continued research into the regulatory mechanisms of PKA and ERK for PRGs will help us better understand the molecular mechanisms underlying the dual effects of PTH, thereby optimizing the current therapeutic use of PTH for osteoporosis.

The role of Ca2+-activated Cl- current (ICl(Ca)) in cardiac arrhythmias is still controversial. It can generate delayed afterdepolarizations in Ca2+-overloaded cells while in other studies incidence of early afterdepolarization (EAD) was reduced by ICl(Ca). Therefore our goal was to examine the role of ICl(Ca) in spatial and temporal heterogeneity of cardiac repolarization and EAD formation. Experiments were performed on isolated canine cardiomyocytes originating from various regions of the left ventricle; subepicardial, midmyocardial and subendocardial cells, as well as apical and basal cells of the midmyocardium. ICl(Ca) was blocked by 0.5mmol/L 9-anthracene carboxylic acid (9-AC). Action potential (AP) changes were tested with sharp microelectrode recording. Whole-cell 9-AC-sensitive current was measured with either square pulse voltage-clamp or AP voltage-clamp (APVC). Protein expression of TMEM16A and Bestrophin-3, ion channel proteins mediating ICl(Ca), was detected by Western blot. 9-AC reduced phase-1 repolarization in every tested cell. 9-AC also increased AP duration in a reverse rate-dependent manner in all cell types except for subepicardial cells. Neither ICl(Ca) density recorded with square pulses nor the normalized expressions of TMEM16A and Bestrophin-3 proteins differed significantly among the examined groups of cells. The early outward component of ICl(Ca) was significantly larger in subepicardial than in subendocardial cells in APVC setting. Applying a typical subepicardial AP as a command pulse resulted in a significantly larger early outward component in both subepicardial and subendocardial cells, compared to experiments when a typical subendocardial AP was applied. Inhibiting ICl(Ca) by 9-AC generated EADs at low stimulation rates and their incidence increased upon beta-adrenergic stimulation. 9-AC increased the short-term variability of repolarization also. We suggest a protective role for ICl(Ca) against risk of arrhythmias by reducing spatial and temporal heterogeneity of cardiac repolarization and EAD formation.

Cancer cells exhibit increased use of nutrients, including glucose and glutamine, to support the bioenergetic and biosynthetic demands of proliferation. We tested the small-molecule inhibitor of glutaminase CB-839 in combination with erlotinib on epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) as a therapeutic strategy to simultaneously impair cancer glucose and glutamine utilization and thereby suppress tumor growth. Here, we show that CB-839 cooperates with erlotinib to drive energetic stress and activate the AMP-activated protein kinase (AMPK) pathway in EGFR (del19) lung tumors. Tumor cells undergo metabolic crisis and cell death, resulting in rapid tumor regression in vivo in mouse NSCLC xenografts. Consistently, positron emission tomography (PET) imaging with 18F-fluoro-2-deoxyglucose (18F-FDG) and 11C-glutamine (11C-Gln) of xenografts indicated reduced glucose and glutamine uptake in tumors following treatment with CB-839 + erlotinib. Therefore, PET imaging with 18F-FDG and 11C-Gln tracers can be used to non-invasively measure metabolic response to CB-839 and erlotinib combination therapy.

BackgroundChemotherapy with docetaxel (Doc) remains the standard treatment for metastatic and castration-resistance prostate cancer (CRPC). However, the clinical success of Doc is limited by its chemoresistance and side effects. This study investigated whether natural products green tea (GT) and quercetin (Q) enhance the therapeutic efficacy of Doc in CRPC in mouse models.MethodsMale severe combined immunodeficiency (SCID) mice (n = 10 per group) were inoculated with androgen-independent prostate cancer PC-3 cells subcutaneously. When tumors were established the intervention started. Mice were administered with GT + Q, Doc 5 mg/kg (LD), GT + Q + LD Doc, Doc 10 mg/kg (HD) or control. The concentration of GT polyphenols in brewed tea administered as drinking water was 0.07% and Q was supplemented in diet at 0.4%. Doc was intravenously injected weekly for 4 weeks, GT and Q given throughout the study.ResultsGT + Q or LD Doc slightly inhibited tumor growth compared to control. However, the combination of GT and Q with LD Doc significantly enhanced the potency of Doc 2-fold and reduced tumor growth by 62% compared to LD Doc in 7-weeks intervention. A decrease of Ki67 and increase of cleaved caspase 7 were observed in tumors by the mixture, along with lowered blood concentrations of growth factors like VEGF and EGF. The mixture significantly elevated the levels of tumor suppressor mir15a and mir330 in tumor tissues. An increased risk of liver toxicity was only observed with HD Doc treatment.ConclusionsThese results provide a promising regimen to enhance the therapeutic effect of Doc in a less toxic manner.

Inactivation of the LKB1 tumor suppressor is a frequent event in non-small cell lung carcinoma (NSCLC) leading to the activation of mTOR complex 1 (mTORC1) and sensitivity to the metabolic stress inducer phenformin. In this study, we explored the combinatorial use of phenformin with the mTOR catalytic kinase inhibitor MLN0128 as a treatment strategy for NSCLC bearing comutations in the LKB1 and KRAS genes. NSCLC is a genetically and pathologically heterogeneous disease, giving rise to lung tumors of varying histologies that include adenocarcinomas and squamous cell carcinomas (SCC). We demonstrate that phenformin in combination with MLN0128 induced a significant therapeutic response in KRAS/LKB1-mutant human cell lines and genetically engineered mouse models of NSCLC that develop both adenocarcinomas and SCCs. Specifically, we found that KRAS/LKB1-mutant lung adenocarcinomas responded strongly to phenformin + MLN0128 treatment, but the response of SCCs to single or combined treatment with MLN0128 was more attenuated due to acquired resistance to mTOR inhibition through modulation of the AKT-GSK signaling axis. Combinatorial use of the mTOR inhibitor and AKT inhibitor MK2206 robustly inhibited the growth and viability of squamous lung tumors, thus providing an effective strategy to overcome resistance. Taken together, our findings define new personalized therapeutic strategies that may be rapidly translated into clinical use for the treatment of KRAS/LKB1-mutant adenocarcinomas and squamous cell tumors.

Growing evidence suggests that tumor-associated macrophages (TAM) promote cancer progression and therapeutic resistance by enhancing angiogenesis, matrix-remodeling, and immunosuppression. In this study, prostate cancer under androgen blockade therapy (ABT) was investigated, demonstrating that TAMs contribute to prostate cancer disease recurrence through paracrine signaling processes. ABT induced the tumor cells to express macrophage colony-stimulating factor 1 (M-CSF1 or CSF1) and other cytokines that recruit and modulate macrophages, causing a significant increase in TAM infiltration. Inhibitors of CSF1 signaling through its receptor, CSF1R, were tested in combination with ABT, demonstrating that blockade of TAM influx in this setting disrupts tumor promotion and sustains a more durable therapeutic response compared with ABT alone.

UnlabelledPharmacologic and global gene deletion studies demonstrate that cyclooxygenase-2 (PTGS2/COX-2) plays a critical role in DMBA/TPA-induced skin tumor induction. Although many cell types in the tumor microenvironment express COX-2, the cell types in which COX-2 expression is required for tumor promotion are not clearly established. Here, cell type-specific Cox-2 gene deletion reveals a vital role for skin epithelial cell COX-2 expression in DMBA/TPA tumor induction. In contrast, myeloid Cox-2 gene deletion has no effect on DMBA/TPA tumorigenesis. The infrequent, small tumors that develop on mice with an epithelial cell-specific Cox-2 gene deletion have decreased proliferation and increased cell differentiation properties. Blood vessel density is reduced in tumors with an epithelial cell-specific Cox-2 gene deletion, compared with littermate control tumors, suggesting a reciprocal relationship in tumor progression between COX-2-expressing tumor epithelial cells and microenvironment endothelial cells. Lipidomics analysis of skin and tumors from DMBA/TPA-treated mice suggests that the prostaglandins PGE2 and PGF2α are likely candidates for the epithelial cell COX-2-dependent eicosanoids that mediate tumor progression. This study both illustrates the value of cell type-specific gene deletions in understanding the cellular roles of signal-generating pathways in complex microenvironments and emphasizes the benefit of a systems-based lipidomic analysis approach to identify candidate lipid mediators of biologic responses.ImplicationsCox-2 gene deletion demonstrates that intrinsic COX-2 expression in initiated keratinocytes is a principal driver of skin carcinogenesis; lipidomic analysis identifies likely prostanoid effectors.

In human tumors, and in mouse models, cyclooxygenase-2 (COX-2) levels are frequently correlated with tumor development/burden. In addition to intrinsic tumor cell expression, COX-2 is often present in fibroblasts, myofibroblasts and endothelial cells of the tumor microenvironment, and in infiltrating immune cells. Intrinsic cancer cell COX-2 expression is postulated as only one of many sources for prostanoids required for tumor promotion/progression. Although both COX-2 inhibition and global Cox-2 gene deletion ameliorate ultraviolet B (UVB)-induced SKH-1 mouse skin tumorigenesis, neither manipulation can elucidate the cell type(s) in which COX-2 expression is required for tumorigenesis; both eliminate COX-2 activity in all cells. To address this question, we created Cox-2(flox/flox) mice, in which the Cox-2 gene can be eliminated in a cell-type-specific fashion by targeted Cre recombinase expression. Cox-2 deletion in skin epithelial cells of SKH-1 Cox-2(flox/flox);K14Cre(+) mice resulted, following UVB irradiation, in reduced skin hyperplasia and increased apoptosis. Targeted epithelial cell Cox-2 deletion also resulted in reduced tumor incidence, frequency, size and proliferation rate, altered tumor cell differentiation and reduced tumor vascularization. Moreover, Cox-2(flox/flox);K14Cre(+) papillomas did not progress to squamous cell carcinomas. In contrast, Cox-2 deletion in SKH-1 Cox-2(flox/flox); LysMCre(+) myeloid cells had no effect on UVB tumor induction. We conclude that (i) intrinsic epithelial COX-2 activity plays a major role in UVB-induced skin cancer, (ii) macrophage/myeloid COX-2 plays no role in UVB-induced skin cancer and (iii) either there may be another COX-2-dependent prostanoid source(s) that drives UVB skin tumor induction or there may exist a COX-2-independent pathway(s) to UVB-induced skin cancer.

The chemopreventive activity of green tea (GT) is limited by the low bioavailability and extensive methylation of GT polyphenols (GTPs) in vivo. We determined whether a methylation inhibitor quercetin (Q) will enhance the chemoprevention of prostate cancer in vivo. Androgen-sensitive LAPC-4 prostate cancer cells were injected subcutaneously into severe combined immunodeficiency (SCID) mice one week before the intervention. The concentration of GTPs in brewed tea administered as drinking water was 0.07% and Q was supplemented in diet at 0.2% or 0.4%. After 6-weeks of intervention tumor growth was inhibited by 3% (0.2% Q), 15% (0.4% Q), 21% (GT), 28% (GT+0.2% Q) and 45% (GT+0.4% Q) compared to control. The concentration of non-methylated GTPs was significantly increased in tumor tissue with GT+0.4% Q treatment compared to GT alone, and was associated with a decreased protein expression of catechol-O-methyltransferase and multidrug resistance-associated protein (MRP)-1. The combination treatment was also associated with a significant increase in the inhibition of proliferation, androgen receptor and phosphatidylinositol 3-kinase/Akt signaling, and stimulation of apoptosis. The combined effect of GT+0.4% Q on tumor inhibition was further confirmed in another experiment where the intervention started prior to tumor inoculation. These results provide a novel regimen by combining GT and Q to improve chemoprevention in a non-toxic manner and warrant future studies in humans.