Acid-sensing receptor GPR4 plays a crucial role in lymphatic cancer metastasis.

Cancer tissues exhibit an acidic microenvironment owing to the accumulation of protons and lactic acid produced by cancer and inflammatory cells. To examine the role of an acidic microenvironment in lymphatic cancer metastasis, gene expression profiling was conducted using human dermal lymphatic endothelial cells (HDLECs) treated with a low pH medium. Microarray and gene set enrichment analysis revealed that acid treatment induced the expression of inflammation-related genes in HDLECs, including genes encoding chemokines and adhesion molecules. Acid treatment-induced chemokines C-X3-C motif chemokine ligand 1 (CX3CL1) and C-X-C motif chemokine ligand 6 (CXCL6) autocrinally promoted the growth and tube formation of HDLECs. The expression of vascular cell adhesion molecule 1 (VCAM-1) increased in HDLECs after acid treatment in a time-dependent manner, which, in turn, enhanced their adhesion to melanoma cells. Among various acid-sensing receptors, HDLECs basally expressed G protein-coupled receptor 4 (GPR4), which was augmented under the acidic microenvironment. The induction of chemokines or VCAM-1 under acidic conditions was attenuated by GPR4 knockdown in HDLECs. In addition, lymph node metastases in a mouse melanoma model were suppressed by administering an anti-VCAM-1 antibody or a GPR4 antagonist. These results suggest that an acidic microenvironment modifies the function of lymphatic endothelial cells via GPR4, thereby promoting lymphatic cancer metastasis. Acid-sensing receptors and their downstream molecules might serve as preventive or therapeutic targets in cancer.

The acid-sensing nociceptor TRPV1 controls breast cancer progression in bone via regulating HGF secretion from sensory neurons.

Cancers showing excessive innervation of sensory neurons (SN) in their microenvironments are associated with poor outcomes due to promoted growth, increased tumor recurrence, metastasis, and cancer pain, suggesting SNs play a regulatory role in cancer aggressiveness. Using a preclinical model in which mouse 4T1 breast cancer (BC) cells were injected into the bone marrow of tibiae, we found 4T1 BC cells aggressively colonized bone with bone destruction and subsequently spread to the lung. Of note, 4T1 BC colonization induced the acidic tumor microenvironment in bone in which SNs showed increased innervation and excitation with elevated expression of the acid-sensing nociceptor transient receptor potential vanilloid-1 (TRPV1), eliciting bone pain (BP) assessed by mechanical hypersensitivity. Further, these excited SNs produced increased hepatocyte growth factor (HGF). Importantly, the administration of synthetic and natural TRPV1 antagonists and genetic deletion of TRPV1 decreased HGF production in SNs and inhibited 4T1 BC colonization in bone, pulmonary metastasis from bone, and BP induction. Our results suggest the TRPV1 of SNs promotes BC colonization in bone and lung metastasis via up-regulating HGF production in SNs. The SN TRPV1 may be a novel therapeutic target for BC growing in the acidic bone microenvironment and for BP.

Dmrt2 promotes transition of endochondral bone formation by linking Sox9 and Runx2.

Endochondral bone formation is fundamental for skeletal development. During this process, chondrocytes undergo multiple steps of differentiation and coordinated transition from a proliferating to a hypertrophic stage, which is critical to advance skeletal development. Here, we identified the transcription factor Dmrt2 (double-sex and mab-3 related transcription factor 2) as a Sox9-inducible gene that promotes chondrocyte hypertrophy in pre-hypertrophic chondrocytes. Epigenetic analysis further demonstrated that Sox9 regulates Dmrt2 expression through an active enhancer located 18 kb upstream of the Dmrt2 gene and that this enhancer's chromatin status is progressively activated through chondrocyte differentiation. Dmrt2-knockout mice exhibited a dwarf phenotype with delayed initiation of chondrocyte hypertrophy. Dmrt2 augmented hypertrophic chondrocyte gene expression including Ihh through physical and functional interaction with Runx2. Furthermore, Dmrt2 deficiency reduced Runx2-dependent Ihh expression. Our findings suggest that Dmrt2 is critical for sequential chondrocyte differentiation during endochondral bone formation and coordinates the transcriptional network between Sox9 and Runx2.

Acidic microenvironment induction of interleukin-8 expression and matrix metalloproteinase-2/-9 activation via acid-sensing ion channel 1 promotes breast cancer cell progression.

The cancer microenvironment exhibits local acidosis compared with the surrounding normal tissue. Many reports have shown that acidosis accelerates the invasiveness and metastasis of cancer, yet the underlying molecular mechanisms remain unclear. In the present study, we focused on acid-induced functional changes through acid receptors in breast cancer cells. Acidic treatment induced interleukin (IL)-8 expression in MDA-MB-231 cells and promoted cell migration and invasion. The acidic microenvironment elevated matrix metalloproteinase (MMP)-2 and MMP-9 activity, and addition of IL-8 had similar effects. However, inhibition of IL-8 suppressed the acid-induced migration and invasion of MDA-MB-231 cells. MDA-MB-231 cells express various acid receptors including ion channels and G protein-coupled receptors. Interestingly, acidic stimulation increased the expression of acid-sensing ion channel 1 (ASIC1), and acid-induced IL-8 was significantly decreased by ASIC1 knockdown. Moreover, phosphorylation of nuclear factor (NF)-κB was induced by acidic treatment, and inhibition of NF-κB activation reduced acid-induced IL-8 expression. These results suggest that IL-8 induction by an acidic microenvironment promotes breast cancer development and that ASIC1 might be a novel therapeutic target for breast cancer metastasis.

The Gain-of-Function Mutation p53R248W Suppresses Cell Proliferation and Invasion of Oral Squamous Cell Carcinoma through the Down-Regulation of Keratin 17.

Keratin 17 (KRT17) expression promotes the proliferation and invasion of oral squamous cell carcinoma (OSCC), and mutations in TP53 have been reported in 65% to 85% of OSCC cases. We studied the correlation between KRT17 expression and TP53 mutants. Ca9-22 cells, which exhibit low KRT17 expression, carried mutant p53 (p53R248W) and p53R248W knockdown promoted KRT17 expression. p53R248W knockdown in Ca9-22 cells promoted migration and invasion activity. In contrast, in HSC3 cells, which have p53 nonsense mutations and exhibit high KRT17 expression, the overexpression of p53R248W decreased KRT17 expression, cell size, proliferation, and migration and invasion activities. In addition, p53R248W significantly suppressed MMP2 mRNA expression and enzyme activity. Moreover, s.c. and orthotopic xenografts were generated from p53R248W- or p53R248Q-expressing HSC3 cells. Tumors formed from p53R248W-expressing HSC3 cells grew more slowly and had a lower Ki-67 index than those derived from the control or p53R248Q-expressing HSC3 cells. Finally, the survival rate of the mice inoculated with p53R248W-expressing HSC3 cells was significantly higher than that of the control mice. These results indicate that the p53R248W mutant suppresses proliferation and invasion activity through the suppression of KRT17 expression. We propose that OSCC with p53R248W-expressing cells may be classified as a new OSCC type that has a good prognosis.

Epithelial Cell-specific Deletion of Microsomal Prostaglandin E Synthase-1 Does Not Influence Colon Tumor Development in Mice.

Activation of the COX-2/microsomal prostaglandin E synthase-1 (mPGES-1)/prostaglandin E2 (PGE2) signaling axis is a hallmark of many cancers, including colorectal cancer, prompting the implementation of prevention strategies targeting COX-2 activity. We have previously shown that targeting the downstream terminal PGE2 synthase, mPGES-1 (Ptges), specifically reduces inducible PGE2 formation without disrupting synthesis of other essential prostanoids, thereby conferring dramatic cancer protection against colon carcinogenesis in multiple mouse models. In order to accelerate its development as a viable drug target, and to better understand the mechanisms by which PGE2 influences colon carcinogenesis, we recently developed a conditional Ptges knockout mouse model (cKO). To evaluate the functional role of Ptges directly within the colonic epithelia, cKO mice were crossed with carbonic anhydrase 1 (Car1)-Cre mice (cKO.Car1), and colon tumors were induced using the azoxymethane/dextran sodium sulfate protocol. Unexpectedly, epithelial-specific blockade of Ptges failed to protect mice against colon tumor development. Further studies using the cKO mouse model will be necessary to pinpoint the cell type-specific location of mPGES-1 and its control of inducible PGE2 formation that drives tumor formation in the colon.

Haploinsufficiency of Casitas B-Lineage Lymphoma Augments the Progression of Colon Cancer in the Background of Adenomatous Polyposis Coli Inactivation.

Casitas B-lineage lymphoma (c-Cbl) is a recently identified ubiquitin ligase of nuclear ß-catenin and a suppressor of colorectal cancer (CRC) growth in cell culture and mouse tumor xenografts. We hypothesized that reduction in c-Cbl in colonic epithelium is likely to increase the levels of nuclear ß-catenin in the intestinal crypt, augmenting CRC tumorigenesis in an adenomatous polyposis coli (APCΔ14/+) mouse model. Haploinsufficient c-Cbl mice (APCΔ14/+ c-Cbl+/-) displayed a significant (threefold) increase in atypical hyperplasia and adenocarcinomas in the small and large intestines; however, no differences were noted in the adenoma frequency. In contrast to the APCΔ14/+ c-Cbl+/+ mice, APCΔ14/+ c-Cbl+/- crypts showed nuclear ß-catenin throughout the length of the crypts and up-regulation of Axin2, a canonical Wnt target gene, and SRY-box transcription factor 9, a marker of intestinal stem cells. In contrast, haploinsufficiency of c-Cbl+/- alone was insufficient to induce tumorigenesis regardless of an increase in the number of intestinal epithelial cells with nuclear ß-catenin and SRY-box transcription factor 9 in APC+/+ c-Cbl+/- mice. This study demonstrates that haploinsufficiency of c-Cbl results in Wnt hyperactivation in intestinal crypts and accelerates CRC progression to adenocarcinoma in the milieu of APCΔ14/+, a phenomenon not found with wild-type APC. While emphasizing the role of APC as a gatekeeper in CRC, this study also demonstrates that combined partial loss of c-Cbl and inactivation of APC significantly contribute to CRC tumorigenesis.

Colon Cancer Prevention with Walnuts: A Longitudinal Study in Mice from the Perspective of a Gut Enterotype-like Cluster.

There is limited understanding of how walnut consumption inhibits the development of colorectal cancer. A possible mechanism may involve alterations to the gut microbiota. In this study, the effects of walnut on gut microbiota were tested in a mouse tumor bioassay using the colonotropic carcinogen, azoxymethane (AOM) added to the total Western diet (TWD). 16S rRNA pyrosequencing identified three enterotype-like clusters (E1, E2, and E3) in this murine model. E1, E2, and E3 are associated with AOM exposure, walnut consumption, and TWD diet, respectively. E2 and E3 showed distinct taxonomic and functional characteristics, while E1 represented an intermediate state. At the family level, E1 and E3 were both enriched with Bacteroidaceae, but driven by two different operational taxonomic units (OTU; OTU-2 for E1, OTU-4 for E3). E2 was overrepresented with Porphyromonadaceae and Lachnospiraceae, with OTU-3 (family Porphyromonadaceae) as the "driver" OTU for this cluster. Functionally, E3 is overrepresented with genes of glycan biosynthesis and metabolism, xenobiotic metabolism, and lipid metabolism. E2 is enriched with genes associated with cell motility, replication and repair, and amino acid metabolism. Longitudinally, E2 represents the gut microbial status of early life in these mice. In comparison with E1 and E3, E2 is associated with a moderate lower tumor burden (P = 0.12). Our results suggest that walnuts may reduce the risk of colorectal cancer within a Western diet by altering the gut microbiota. Our findings provide further evidence that colorectal cancer risk is potentially modifiable by diet via alterations to the microbiota.

Dietary Walnut Supplementation Alters Mucosal Metabolite Profiles During DSS-Induced Colonic Ulceration.

Walnuts contain a complex array of natural compounds and phytochemicals that exhibit a wide range of health benefits, including protection against inflammation and colon cancer. In this study, we assess the effects of dietary supplementation with walnuts on colonic mucosal injury induced in mice by the ulcerogenic agent, dextran sodium sulfate (DSS). C57Bl/6J mice were started on the Total Western Diet supplemented with freshly-ground whole walnuts (0, 3.5, 7 and 14% g/kg) 2 weeks prior to a 5-day DSS treatment and walnut diets were continued throughout the entire experimental period. Mice were examined at 2 days or 10 days after withdrawal of DSS. In a separate study, a discovery-based metabolite profiling analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed on fecal samples and colonic mucosa following two weeks of walnut supplementation. Dietary walnut supplementation showed significant effects in the 10-day post-DSS recovery-phase study, in which the extent of ulceration was significantly reduced (7.5% vs. 0.3%, p < 0.05) with 14% walnuts. In the metabolite-profiling analysis, walnuts caused a significant increase in several polyunsaturated fatty acids (PUFAs), including docosahexaenoic acid (DHA) and 9-oxo-10(E),12(E)-octadecadienoic acid (9-oxoODA), as well as kynurenic acid. In colon tissue samples, walnuts caused a significant increase in the levels of S-adenosylhomocysteine (SAH) and betaine, important components of fatty acid ß-oxidation. These metabolite changes may contribute in part to the observed protection against DSS-induced inflammatory tissue injury.

Dietary Walnuts Protect Against Obesity-Driven Intestinal Stem Cell Decline and Tumorigenesis.

Obesity can negatively impact intestinal homeostasis, and increase colon cancer risk and related mortality. Thus, given the alarmingly high rates of obesity in the US and globally, it is critical to identify practical strategies that can break the obesity-cancer link. Walnuts have been increasingly recognized to mitigate cancer risk, and contain many bioactive constituents with antioxidant and anti-inflammatory properties that could potentially counteract pathways thought to be initiators of obesity-related cancer. Therefore, the purpose of this study was to determine if walnuts could preserve intestinal homeostasis, and attenuate tumorigenesis and growth in the context of obesity and a high calorie diet. To this end, we studied effects of walnuts on these parameters under different dietary conditions in wildtype mice, two independent Apc models (Apc1638N/+ and ApcΔ14), and in MC38 colon cancer cells in vivo, respectively. Walnuts did not alter the metabolic phenotype or intestinal morphology in normal mice fed either a low-fat diet (LFD), LFD with 6% walnuts (LFD+W), high-fat diet (HFD), or HFD with 7.6% walnuts (HFD+W). However, walnuts did lead to a significant reduction in circulating CCL5 and preserved intestinal stem cell (ISC) function under HFD-fed conditions. Furthermore, walnuts reduced tumor multiplicity in Apc1638N/+ male HFD+W animals, as compared to HFD controls (3.7 ± 0.5 vs. 2.5 ± 0.3; P = 0.015), tended to reduce the number of adenocarcinomas (0.67 ± 0.16 vs. 0.29 ± 0.12; P = 0.07), and preferentially limited tumor growth in ApcΔ14 male mice (P = 0.019) fed a high-calorie western-style diet. In summary, these data demonstrate that walnuts confer significant protection against intestinal tumorigenesis and growth and preserve ISC function in the context of a high-calorie diet and obesity. Thus, these data add to the accumulating evidence connecting walnuts as a potentially effective dietary strategy to break the obesity-colon cancer link.

Spindle Assembly Disruption and Cancer Cell Apoptosis with a CLTC-Binding Compound.

AK3 compounds are mitotic arrest agents that induce high levels of γH2AX during mitosis and apoptosis following release from arrest. We synthesized a potent AK3 derivative, AK306, that induced arrest and apoptosis of the HCT116 colon cancer cell line with an EC50 of approximately 50 nmol/L. AK306 was active on a broad spectrum of cancer cell lines with total growth inhibition values ranging from approximately 25 nmol/L to 25 µmol/L. Using biotin and BODIPY-linked derivatives of AK306, binding to clathrin heavy chain (CLTC/CHC) was observed, a protein with roles in endocytosis and mitosis. AK306 inhibited mitosis and endocytosis, while disrupting CHC cellular localization. Cells arrested in mitosis by AK306 showed the formation of multiple microtubule-organizing centers consisting of pericentrin, γ-tubulin, and Aurora A foci, without apparent centrosome amplification. Cells released from AK306 arrest were unable to form bipolar spindles, unlike nocodazole-released cells that reformed spindles and completed division. Like AK306, CHC siRNA knockdown disrupted spindle formation and activated p53. A short-term (3-day) treatment of tumor-bearing APC-mutant mice with AK306 increased apoptosis in tumors, but not normal mucosa. These findings indicate that targeting the mitotic CHC complex can selectively induce apoptosis and may have therapeutic value.Implication: Disruption of clathrin with a small-molecule inhibitor, AK306, selectively induces apoptosis in cancer cells by disrupting bipolar spindle formation. Mol Cancer Res; 16(9); 1361-72. ©2018 AACR.

Cyclooxygenase-1 and -2 Play Contrasting Roles in Listeria-Stimulated Immunity.

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenase (COX) activity and are commonly used for pain relief and fever reduction. NSAIDs are used following childhood vaccinations and cancer immunotherapies; however, how NSAIDs influence the development of immunity following these therapies is unknown. We hypothesized that NSAIDs would modulate the development of an immune response to Listeria monocytogenes-based immunotherapy. Treatment of mice with the nonspecific COX inhibitor indomethacin impaired the generation of cell-mediated immunity. This phenotype was due to inhibition of the inducible COX-2 enzyme, as treatment with the COX-2-selective inhibitor celecoxib similarly inhibited the development of immunity. In contrast, loss of COX-1 activity improved immunity to L. monocytogenes Impairments in immunity were independent of bacterial burden, dendritic cell costimulation, or innate immune cell infiltrate. Instead, we observed that PGE2 production following L. monocytogenes is critical for the formation of an Ag-specific CD8+ T cell response. Use of the alternative analgesic acetaminophen did not impair immunity. Taken together, our results suggest that COX-2 is necessary for optimal CD8+ T cell responses to L. monocytogenes, whereas COX-1 is detrimental. Use of pharmacotherapies that spare COX-2 activity and the production of PGE2 like acetaminophen will be critical for the generation of optimal antitumor responses using L. monocytogenes.

Impact of heart-specific disruption of the circadian clock on systemic glucose metabolism in mice.

The daily rhythm of glucose metabolism is governed by the circadian clock, which consists of cell-autonomous clock machineries residing in nearly every tissue in the body. Disruption of these clock machineries either environmentally or genetically induces the dysregulation of glucose metabolism. Although the roles of clock machineries in the regulation of glucose metabolism have been uncovered in major metabolic tissues, such as the pancreas, liver, and skeletal muscle, it remains unknown whether clock function in non-major metabolic tissues also affects systemic glucose metabolism. Here, we tested the hypothesis that disruption of the clock machinery in the heart might also affect systemic glucose metabolism, because heart function is known to be associated with glucose tolerance. We examined glucose and insulin tolerance as well as heart phenotypes in mice with heart-specific deletion of Bmal1, a core clock gene. Bmal1 deletion in the heart not only decreased heart function but also led to systemic insulin resistance. Moreover, hyperglycemia was induced with age. Furthermore, heart-specific Bmal1-deficient mice exhibited decreased insulin-induced phosphorylation of Akt in the liver, thus indicating that Bmal1 deletion in the heart causes hepatic insulin resistance. Our findings revealed an unexpected effect of the function of clock machinery in a non-major metabolic tissue, the heart, on systemic glucose metabolism in mammals.

A novel bioactive derivative of eicosapentaenoic acid (EPA) suppresses intestinal tumor development in ApcΔ14/+ mice.

Familial adenomatous polyposis (FAP) is a genetic disorder characterized by the development of hundreds of polyps throughout the colon. Without prophylactic colectomy, most individuals with FAP develop colorectal cancer at an early age. Treatment with EPA in the free fatty acid form (EPA-FFA) has been shown to reduce polyp burden in FAP patients. Since high-purity EPA-FFA is subject to rapid oxidation, a stable form of EPA compound has been developed in the form of magnesium l-lysinate bis-eicosapentaenoate (TP-252). We assessed the chemopreventive efficacy of TP-252 on intestinal tumor formation using ApcΔ14/+ mice and compared it with EPA-FFA. TP-252 was supplemented in a modified AIN-93G diet at 1, 2 or 4% and EPA-FFA at 2.5% by weight and administered to mice for 11 weeks. We found that administration of TP-252 significantly reduced tumor number and size in the small intestine and colon in a dose-related manner and as effectively as EPA-FFA. To gain further insight into the cancer protection afforded to the colon, we performed a comprehensive lipidomic analysis of total fatty acid composition and eicosanoid metabolites. Treatment with TP-252 significantly decreased the levels of arachidonic acid (AA) and increased EPA concentrations within the colonic mucosa. Furthermore, a classification and regression tree (CART) analysis revealed that a subset of fatty acids, including EPA and docosahexaenoic acid (DHA), and their downstream metabolites, including PGE3 and 14-hydroxy-docosahexaenoic acid (HDoHE), were strongly associated with antineoplastic activity. These results indicate that TP-252 warrants further clinical development as a potential strategy for delaying colectomy in adolescent FAP patients.

Incidence of pancreatic cancer is dramatically increased by a high fat, high calorie diet in KrasG12D mice.

Epidemiologic data has linked obesity to a higher risk of pancreatic cancer, but the underlying mechanisms are poorly understood. To allow for detailed mechanistic studies in a relevant model mimicking diet-induced obesity and pancreatic cancer, a high-fat, high-calorie diet (HFCD) was given to P48+/Cre;LSL-KRASG12D (KC) mice carrying a pancreas-specific oncogenic Kras mutation. The mice were randomly allocated to a HFCD or control diet (CD). Cohorts were sacrificed at 3, 6, and 9 months and tissues were harvested for further analysis. Compared to CD-fed mice, HFCD-fed animals gained significantly more weight. Importantly, the cancer incidence was remarkably increased in HFCD-fed KC mice, particularly in male KC mice. In addition, KC mice fed the HFCD showed more extensive inflammation and fibrosis, and more advanced PanIN lesions in the pancreas, compared to age-matched CD-fed animals. Interestingly, we found that the HFCD reduced autophagic flux in PanIN lesions in KC mice. Further, exome sequencing of isolated murine PanIN lesions identified numerous genetic variants unique to the HFCD. These data underscore the role of sustained inflammation and dysregulated autophagy in diet-induced pancreatic cancer development and suggest that diet-induced genetic alterations may contribute to this process. Our findings provide a better understanding of the mechanisms underlying the obesity-cancer link in males and females, and will facilitate the development of interventions targeting obesity-associated pancreatic cancer.

An Autopsy Case of Fulminant Amebic Colitis in a Patient with a History of Rheumatoid Arthritis.

Generally, amebic colitis is localized around the mucosal membrane and often accompanied by diarrhea and abdominal pain. We describe a patient with a history of rheumatoid arthritis who had received prolonged steroid therapy. The patient complained of breathing difficulties because of rheumatoid lung disease. Although the patient was given antibacterial agent, the symptoms did not improve until death. We did an autopsy and found that he had fulminant amebic colitis, although the patient was not previously examined. Histochemical analysis revealed severe inflammation and full-thickness necrosis of the colon by ameba, suggesting the involvement of ameba in the progression of the overall condition.

Acidic microenvironments induce lymphangiogenesis and IL-8 production via TRPV1 activation in human lymphatic endothelial cells.

Local acidosis is one of the characteristic features of the cancer microenvironment. Many reports indicate that acidosis accelerates the proliferation and invasiveness of cancer cells. However, whether acidic conditions affect lymphatic metastasis is currently unknown. In the present study, we focused on the effects of acidosis on lymphatic endothelial cells (LECs) to assess the relationship between acidic microenvironments and lymph node metastasis. We demonstrated that normal human LECs express various acid receptors by immunohistochemistry and reverse transcriptase-polymerase chain reaction (PCR). Acidic stimulation with low pH medium induced morphological changes in LECs to a spindle shape, and significantly promoted cellular growth and tube formation. Moreover, real-time PCR revealed that acidic conditions increased the mRNA expression of interleukin (IL)-8. Acidic stimulation increased IL-8 production in LECs, whereas a selective transient receptor potential vanilloid subtype 1 (TRPV1) antagonist, 5'-iodoresiniferatoxin, decreased IL-8 production. IL-8 accelerated the proliferation of LECs, and inhibition of IL-8 diminished tube formation and cell migration. In addition, phosphorylation of nuclear factor (NF)-κB was induced by acidic conditions, and inhibition of NF-κB activation reduced acid-induced IL-8 expression. These results suggest that acidic microenvironments in tumors induce lymphangiogenesis via TRPV1 activation in LECs, which in turn may promote lymphatic metastasis.

Effects of Walnut Consumption on Colon Carcinogenesis and Microbial Community Structure.

Walnuts are composed of a complex array of biologically active constituents with individual cancer-protective properties. Here, we assessed the potential benefit of whole walnut consumption in a mouse tumor bioassay using azoxymethane. In study 1, a modest reduction (1.3-fold) in tumor numbers was observed in mice fed a standard diet (AIN-76A) containing 9.4% walnuts (15% of total fat). In study 2, the effects of walnut supplementation was tested in the Total Western Diet (TWD). There was a significant reduction (2.3-fold; P < 0.02) in tumor numbers in male mice fed TWD containing 7% walnuts (10.5% of total fat). Higher concentrations of walnuts lacked inhibitory effects, particularly in female mice, indicating there may be optimal levels of dietary walnut intake for cancer prevention. Since components of the Mediterranean diet have been shown to affect the gut microbiome, the effects of walnuts were therefore tested in fecal samples using 16S rRNA gene sequencing. Carcinogen treatment reduced the diversity and richness of the gut microbiome, especially in male mice, which exhibited lower variability and greater sensitivity to environmental changes. Analysis of individual operational taxonomic units (OTU) identified specific groups of bacteria associated with carcinogen exposure, walnut consumption, and/or both variables. Correlation analysis also identified specific OTU clades that were strongly associated with the presence and number of tumors. Taken together, our results indicate that walnuts afford partial protection to the colon against a potent carcinogenic insult, and this may be due, in part, to walnut-induced changes to the gut microbiome. Cancer Prev Res; 9(8); 692-703. ©2016 AACR.

Cellular fibronectin 1 promotes VEGF-C expression, lymphangiogenesis and lymph node metastasis associated with human oral squamous cell carcinoma.

Lymph node metastasis (LNM) is associated with poor survival in patients with oral squamous cell carcinoma (OSCC). Vascular endothelial growth factor-C (VEGF-C) is thought to be responsible for increased lymphangiogenesis and LNM. Understanding of the mechanism by which VEGF-C expression is regulated in OSCC is thus important to design logic therapeutic interventions. We showed that inoculation of the SAS human OSCC cells expressing the venus GFP (V-SAS cells) into the tongue in nude mice developed LNM. V-SAS cells in LNM were isolated by FACS and re-inoculated into the tongue. This procedure was repeated eight times, establishing V-SAS-LM8 cells. Differential metastasis PCR array between the parental V-SAS and V-SAS-LM8 was performed to identify a molecule responsible for lymphangiogenesis and LNM. Fibronectin 1 (FN1) expression was elevated in V-SAS-LM8 cells compared to V-SAS-cells. V-SAS-LM8 tongue tumor showed increased expression of FN1 and VEGF-C, and promoted lymphangiogenesis and LNM compared with V-SAS tumor. Further, phosphorylation of focal adhesion kinase (FAK), a main downstream signaling molecule of FN1, was up-regulated, and epithelial-mesenchymal transition (EMT) was promoted in V-SAS-LM8 cells. Silencing of FN1 by shRNA in V-SAS-LM8 cells decreased FAK phosphorylation, VEGF-C expression and inhibited lymphangiogenesis and LNM. EMT was also reversed. The FAK phosphorylation inhibitor PF573228 also decreased VEGF-C expression and reversed EMT in V-SAS-LM8 cells. Finally, we detected intense FN1 expression in some clinical specimens obtained from OSCC patients with LNM. These results demonstrate that elevated expression of cellular FN1 and following activation of FAK lead to increased VEGF-C expression, lymphangiogenesis and LNM and promoted EMT in SAS human OSCC cells and suggest that FN1-phosphorylated FAK signaling cascade is a potential therapeutic target in the treatment of LNM in OSCC.

Regulation of VDR Expression in Apc-Mutant Mice, Human Colon Cancers and Adenomas.

One variable that may affect the ability of vitamin D to reduce colon cancer risk is the expression of its high-affinity receptor, VDR. Here, we show that vitamin D does not reduce tumor formation in Apc(Δ14/+) mice and that VDR expression is lost in the majority of the colon tumor cells. The extent of VDR loss corresponded inversely to the level of ß-catenin nuclear localization and could be observed in early lesions composed of just a few crypts. Analysis of reported VDR regulators showed that the repressing class I histone deacetylases (HDAC) were significantly elevated in the tumors (up to 4-fold), whereas the VDR-activating retinoid X receptors (RXR) were downregulated (∼50%). Expression of the Slug repressor was also increased, but was found primarily in stromal cells. Analysis of epigenetically active compounds on colon cell lines and intestinal organoids showed that HDAC inhibitors were particularly adept at stimulating VDR expression. Treatment of tumor-bearing Apc(Δ14/+) mice with the HDAC inhibitor panobinostat increased VDR expression in the tumors and normal mucosa. The RXR agonist bexarotene failed to activate VDR expression, indicating that RXR ligands were not limiting. Analysis of human microarray data indicated that VDR mRNA is frequently downregulated in colon adenomas, which correlated positively with RXRA expression and inversely with HDAC 2 and 8 expression. Human adenomas showed variable VDR protein expression levels, both between and within individual lesions. Determining the mechanisms of VDR regulation in colon neoplasms may significantly enhance our ability to use vitamin D as a cancer prevention agent.