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Emerging evidence underscores an association between age-related macular degeneration (AMD) and periodontal disease (PD), yet the biological basis of this linkage and the specific role of oral dysbiosis caused by PD in AMD pathophysiology remains unclear. Furthermore, a simple reproducible model that emulates characteristics of both AMD and PD has been lacking. Hence, we established a novel AMD+PD murine model to decipher the potential role of oral infection (ligature-enhanced) with the keystone periodontal pathogen Porphyromonas gingivalis, in the progression of neovasculogenesis in a laser-induced choroidal-neovascularization (Li-CNV) mouse retina. By a combination of fundus photography, optical coherence tomography, and fluorescein angiography, we documented inflammatory drusen-like lesions, reduced retinal thickness, and increased vascular leakage in AMD+PD mice retinae. H&E further confirmed a significant reduction of retinal thickness and subretinal drusen-like deposits. Immunofluorescence microscopy revealed significant induction of choroidal/retinal vasculogenesis in AMD+PD mice. qPCR identified increased expression of oxidative-stress, angiogenesis, pro-inflammatory mediators, whereas antioxidants and anti-inflammatory genes in AMD+PD mice retinae were notably decreased. Through qPCR, we detected Pg and its fimbrial 16s-RrNA gene expression in the AMD+PD mice retinae. To sum-up, this is the first in vivo study signifying a role of periodontal infection in augmentation of AMD phenotype, with the aid of a pioneering AMD+PD murine model established in our laboratory.

Antibody-mediated protection against pathogens is crucial to a healthy life. However, the recent SARS-CoV-2 pandemic has shown that pre-existing comorbid conditions including kidney disease account for compromised humoral immunity to infections. Individuals with kidney disease are not only susceptible to infections but also exhibit poor vaccine-induced antibody response. Using multiple mouse models of kidney disease, we demonstrate that renal dysfunction inhibits germinal center (GC) response against T-dependent antigens. GC B cells exhibit increased apoptosis in kidney disease. Uremic toxin hippuric acid drives loss of mitochondrial membrane potential, leading to increased apoptosis of GC B cells in a G-protein-coupled receptor 109A dependent manner. Finally, GC B cells and antibody titer are diminished in mice with kidney disease following influenza virus infection, a major cause of mortality in individuals with renal disorders. These results provide a mechanistic understanding of how renal dysfunction suppresses humoral immunity in patients with kidney disease., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

New Ursolic Acid (UA) conjugates were synthesized using optimized synthetic protocols through the molecular hybridization approach at C-3 and C-28. This resulted in the targeted molecules being produced in good yields. Some of the synthesized conjugates showed significantly relevant bioactivity against mammalian cells and in animal models of cancers. Selected UA conjugates were tested against bladder and breast cancer cell lines. The conjugates showed moderate to significantly enhanced antiproliferative activities against Triple Negative Breast Cancer (TNBC; MDA-MB 231), which is an aggressive tumor making up about 10-15 % of all breast cancers and bladder (T24 and 5637) cancer cell lines. These properties were superior to the parent UA. Among all the synthesized compounds, 18 c and 18 d have exhibited promising antiproliferative and cytotoxic properties against all tested cancer cell lines. However, 18 d has proved to be exceptionally selective for cancer cell lines, showing more cytotoxicity towards them than normal epithelial cells (MCF-12A). Compound 18 d has demonstrated cytotoxicity against tumor cells, including those intrinsically resistant to chemotherapy drugs such as 2-difluoro-deoxy cytidine (Gemcitabine). The activity of the UA conjugates on tumor cells was mediated by multiple cytotoxic mechanisms, including drug-induced cytotoxic autophagy and programmed cell death, indicating a novel possibility of combination therapy., (© 2024 Wiley-VCH GmbH.)

The senescence phenotype is heterogeneous, as observed by the context-dependent differential expression of senescence markers. Here, we provide evidence to demonstrate an inverse relationship in the expression pattern of the two murine variants of p21 (p21v1, and p21v2) in aging and hemorrhagic shock. While an upregulation of p21v1 was observed following hemorrhagic shock injury, p21v2 was upregulated in the aged mouse. We further show that the p21v1 response is, at least, partially independent of p53., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Background: Oleanolic acid (OA) and Ursolic acid (UA) are bioactive triterpenoids. Reported activities vary with the dose used for testing their activities in vitro. Studies using doses of ≥20 µM showed apoptosis activities in cancer cells. However, reported drug levels in circulation achieved by oral administration of UA and OA are ≤2 µM, thus limiting their use for treatment or delivering a combination treatment., Materials and Methods: The present report demonstrates the efficacy of OA, UA, and OA + UA on tumor cell-specific cytotoxicity at low doses (5 µM to 10 µM) in breast cancer (BrCa) cell lines MCF7 and MDA-MB231., Results: The data show that both OA and UA killed BrCa cells at low doses, but were significantly less toxic to MCF-12A, a non-tumorigenic cell line. Moreover, OA + UA at ≤10 µM was lethal to BrCa cells. Mechanistic studies unraveled the significant absence of apoptosis, but their cytotoxicity was due to the induction of excessive autophagy at a OA + UA dose of 5 µM each. A link to drug-induced cytotoxic autophagy was established by demonstrating a lack of their cytotoxicity by silencing the autophagy-targeting genes (ATGs), which prevented OA-, UA-, or OA + UA-induced cell death. Further, UA or OA + UA treatment of BrCa cells caused an inhibition of PI3 kinase-mediated phosphorylation of Akt/mTOR, the key pathways that regulate cancer cell survival, metabolism, and proliferation., Discussion: Combinations of a PI3K inhibitor (LY294002) with OA, UA, or OA + UA synergistically inhibited BrCa cell survival. Therefore, the dominance of cytotoxic autophagy by inhibiting PI3K-mediated autophagy may be the primary mechanism of PTT-induced anticancer activity in BrCa cells., Conclusion: These results suggest it would be worthwhile testing combined OA and UA in clinical settings.

Tumor cells produce excessive reactive oxygen species (ROS) but cannot detoxify ROS if they are due to an external agent. An agent that produces toxic levels of ROS, specifically in tumor cells, could be an effective anticancer drug. CMC-2 is a molecular hybrid of the bioactive polyphenol curcumin conjugated to dichloroacetate (DCA) via a glycine bridge. The CMC-2 was tested for its cytotoxic antitumor activities and killed both naïve and multidrug-resistant (MDR) bladder cancer (BCa) cells with equal potency (<1.0 µM); CMC-2 was about 10-15 folds more potent than curcumin or DCA. Growth of human BCa xenograft in mice was reduced by >50% by oral gavage of 50 mg/kg of CMC-2 without recognizable systemic toxicity. Doses that used curcumin or DCA showed minimum antitumor effects. In vitro, the toxicity of CMC-2 in both naïve and MDR cells depended on increased intracellular ROS in tumor cells but not in normal cells at comparable doses. Increased ROS caused the permeabilization of mitochondria and induced apoptosis. Further, adding N-Acetyl cysteine (NAC), a hydroxyl radical scavenger, abolished excessive ROS production and CMC-2's cytotoxicity. The lack of systemic toxicity, equal potency against chemotherapy -naïve and resistant tumors, and oral bioavailability establish the potential of CMC-2 as a potent drug against bladder cancers.

Autosomal-dominant polycystic kidney disease (ADPKD) is a common monogenic disease characterized by the formation of fluid-filled renal cysts, loss of mitochondrial function, decreased fatty acid oxidation, increased glycolysis, and likely renal failure. We previously demonstrated that inducing a state of ketosis ameliorates or reverses PKD progression in multiple animal models. In this study, we compare time-restricted feeding and 48-h periodic fasting regimens in both juvenile and adult Cy/+ rats. Both fasting regimens potently prevent juvenile disease progression and partially reverse PKD in adults. To explore the mechanism of fasting, we administered β-hydroxybutyrate (BHB) to Cy/+ rats and orthologous mouse models of PKD ( Pkd1 RC/RC , Pkd1-Ksp:Cre ). BHB recapitulated the effects of fasting in these models independent of stereoisomer, suggesting the effects of BHB are largely due to its signaling functions. These findings implicate the use of ketogenic metabolic therapy and BHB supplementation as potential disease modifiers of PKD and point toward underlying mechanisms., Competing Interests: J.A.T. and T.W. are partial owners in the Benefit Corporation Santa Barbara Nutrients and are inventors on US patent No. 11,013,705 and International Publication No. WO 2020/186154 A1 for the use of the combination of BHB and citrate in PKD. T.W. was on the scientific advisory board of Chinook Therapeutics and has received research funding from Chinook Therapeutics and Kyowa Kirin, and speaker fees from Otsuka. T.W. was on the scientific advisory board of Chinook Therapeutics., (© 2024 The Author(s).)

Ketogenesis is considered to occur primarily in liver to generate ketones as an alternative energy source for non-hepatic tissues when glucose availability/utilization is impaired. 3-Hydroxy-3-methylglutaryl-CoA synthase-2 (HMGCS2) mediates the rate-limiting step in this mitochondrial pathway. Publicly available databases show marked down-regulation of HMGCS2 in colonic tissues in Crohn's disease and ulcerative colitis. This led us to investigate the expression and function of this pathway in colon and its relevance to colonic inflammation in mice. Hmgcs2 is expressed in cecum and colon. As global deletion of Hmgcs2 showed significant postnatal mortality, we used a conditional knockout mouse with enzyme deletion restricted to intestinal tract. These mice had no postnatal mortality. Fasting blood ketones were lower in these mice, indicating contribution of colonic ketogenesis to circulating ketones. There was also evidence of gut barrier breakdown and increased susceptibility to experimental colitis with associated elevated levels of IL-6, IL-1β, and TNF-α in circulation. Interestingly, many of these phenomena were mostly evident in male mice. Hmgcs2 expression in colon is controlled by colonic microbiota as evidenced from decreased expression in germ-free mice and antibiotic-treated conventional mice and from increased expression in a human colonic epithelial cell line upon treatment with aqueous extracts of cecal contents. Transcriptomic analysis of colonic epithelia from control mice and Hmgcs2-null mice indicated an essential role for colonic ketogenesis in the maintenance of optimal mitochondrial function, cholesterol homeostasis, and cell-cell tight-junction organization. These findings demonstrate a sex-dependent obligatory role for ketogenesis in protection against colonic inflammation in mice., (© 2024 The Author(s).)

The paucity of preclinical models that recapitulate COVID-19 pathology without requiring SARS-COV-2 adaptation and humanized/transgenic mice limits research into new therapeutics against the frequently emerging variants-of-concern. We developed virus-free models by C57BL/6 mice receiving oropharyngeal instillations of a SARS-COV-2 ribo-oligonucleotide common in all variants or specific to Delta/Omicron variants, concurrently with low-dose bleomycin. Mice developed COVID-19-like lung pathologies including ground-glass opacities, interstitial fibrosis, congested alveoli, and became moribund. Lung tissues from these mice and bronchoalveolar lavage and lung tissues from patients with COVID-19 showed elevated levels of hyaluronic acid (HA), HA-family members, an inflammatory signature, and immune cell infiltration. 4-methylumbelliferone (4-MU), an oral drug for biliary-spasm treatment, inhibits HA-synthesis. At the human equivalent dose, 4-MU prevented/inhibited COVID-19-like pathologies and long-term morbidity; 4-MU and metabolites accumulated in mice lungs. Therefore, these versatile SARS-COV-2 ribo-oligonucleotide oropharyngeal models recapitulate COVID-19 pathology, with HA as its critical mediator and 4-MU as a potential therapeutic for COVID-19., Competing Interests: None., (© 2024 The Authors.)

APCs such as dendritic cells and macrophages play a pivotal role in mediating immune tolerance and restoring intestinal immune homeostasis by limiting inflammatory responses against commensal bacteria. However, cell-intrinsic molecular regulators critical for programming intestinal APCs to a regulatory state rather than an inflammatory state are unknown. In this study, we report that the transcription factor retinoid X receptor α (RXRα) signaling in CD11c+ APCs is essential for suppressing intestinal inflammation by imparting an anti-inflammatory phenotype. Using a mouse model of ulcerative colitis, we demonstrated that targeted deletion of RXRα in CD11c+ APCs in mice resulted in the loss of T cell homeostasis with enhanced intestinal inflammation and increased histopathological severity of colonic tissue. This was due to the increased production of proinflammatory cytokines that drive Th1/Th17 responses and decreased expression of immune-regulatory factors that promote regulatory T cell differentiation in the colon. Consistent with these findings, pharmacological activation of the RXRα pathway alleviated colitis severity in mice by suppressing the expression of inflammatory cytokines and limiting Th1/Th17 cell differentiation. These findings identify an essential role for RXRα in APCs in regulating intestinal immune homeostasis and inflammation. Thus, manipulating the RXRα pathway could provide novel opportunities for enhancing regulatory responses and dampening colonic inflammation., (Copyright © 2023 by The American Association of Immunologists, Inc.)

The epidermal growth factor receptor (EGFR) is commonly upregulated in multiple cancer types, including breast cancer. In the present study, evidence is provided in support of the premise that upregulation of the EGFR/MEK1/MAPK1/2 signaling axis during antiestrogen treatment facilitates the escape of breast cancer cells from BimEL‑dependent apoptosis, conferring resistance to therapy. This conclusion is based on the findings that ectopic BimEL cDNA overexpression and confocal imaging studies confirm the pro‑apoptotic role of BimEL in ERα expressing breast cancer cells and that upregulated EGFR/MEK1/MAPK1/2 signaling blocks BimEL pro‑apoptotic action in an antiestrogen‑resistant breast cancer cell model. In addition, the present study identified a pro‑survival role for autophagy in antiestrogen resistance while EGFR inhibitor studies demonstrated that a significant percentage of antiestrogen‑resistant breast cancer cells survive EGFR targeting by pro‑survival autophagy. These pre‑clinical studies establish the possibility that targeting both the MEK1/MAPK1/2 signaling axis and pro‑survival autophagy may be required to eradicate breast cancer cell survival and prevent the development of antiestrogen resistance following hormone treatments. The present study uniquely identified EGFR upregulation as one of the mechanisms breast cancer cells utilize to evade the cytotoxic effects of antiestrogens mediated through BimEL‑dependent apoptosis.

Background: Hyaluronic acid (HA) promotes cancer metastasis; however, the currently approved treatments do not target HA. Metastatic renal carcinoma (mRCC) is an incurable disease. Sorafenib (SF) is a modestly effective antiangiogenic drug for mRCC. Although only endothelial cells express known SF targets, SF is cytotoxic to RCC cells at concentrations higher than the pharmacological-dose (5-µM). Using patient cohorts, mRCC models, and SF combination with 4-methylumbelliferone (MU), we discovered an SF target in RCC cells and targeted it for treatment., Methods: We analyzed HA-synthase (HAS1, HAS2, HAS3) expression in RCC cells and clinical (n = 129), TCGA-KIRC (n = 542), and TCGA-KIRP (n = 291) cohorts. We evaluated the efficacy of SF and SF plus MU combination in RCC cells, HAS3-transfectants, endothelial-RCC co-cultures, and xenografts., Results: RCC cells showed increased HAS3 expression. In the clinical and TCGA-KIRC/TCGA-KIRP cohorts, higher HAS3 levels predicted metastasis and shorter survival. At > 10-µM dose, SF inhibited HAS3/HA-synthesis and RCC cell growth. However, at ≤ 5-µM dose SF in combination with MU inhibited HAS3/HA synthesis, growth of RCC cells and endothelial-RCC co-cultures, and induced apoptosis. The combination inhibited motility/invasion and an HA-signaling-related invasive-signature. We previously showed that MU inhibits SF inactivation in RCC cells. While HAS3-knockdown transfectants were sensitive to SF, ectopic-HAS3-expression induced resistance to the combination. In RCC models, the combination inhibited tumor growth and metastasis with little toxicity; however, ectopic-HAS3-expressing tumors were resistant., Conclusion: HAS3 is the first known target of SF in RCC cells. In combination with MU (human equivalent-dose, 0.6-1.1-g/day), SF targets HAS3 and effectively abrogates mRCC., (© 2022. The Author(s).)

Ursolic acid (UA) is a pentacyclic triterpene isolated from a large variety of vegetables, fruits and many traditional medicinal plants. It is a structural isomer of Oleanolic Acid. The medicinal application of UA has been explored extensively over the last two decades. The diverse pharmacological properties of UA include anti-inflammatory, antimicrobial, antiviral, antioxidant, anti-proliferative, etc. Especially, UA holds a promising position, potentially, as a cancer preventive and therapeutic agent due to its relatively non-toxic properties against normal cells but its antioxidant and antiproliferative activities against cancer cells. Cell culture studies have shown interference of UA with multiple pharmacological and molecular targets that play a critical role in many cells signaling pathways. Although UA is considered a privileged natural product, its clinical applications are limited due to its low absorption through the gastro-intestinal track and rapid elimination. The low bioavailability of UA limits its use as a therapeutic drug. To overcome these drawbacks and utilize the importance of the scaffold, many researchers have been engaged in designing and developing synthetic analogs of UA via structural modifications. This present review summarizes the synthetic UA analogs and their cytotoxic antiproliferative properties reported in the last two decades.

Extraintestinal manifestations are common in inflammatory bowel disease and involve several organs, including the kidney. However, the mechanisms responsible for renal manifestation in inflammatory bowel disease are not known. In this study, we show that the Wnt-lipoprotein receptor-related proteins 5 and 6 (LRP5/6) signaling pathway in macrophages plays a critical role in regulating colitis-associated systemic inflammation and renal injury in a murine dextran sodium sulfate-induced colitis model. Conditional deletion of the Wnt coreceptors LRP5/6 in macrophages in mice results in enhanced susceptibility to dextran sodium sulfate colitis-induced systemic inflammation and acute kidney injury (AKI). Furthermore, our studies show that aggravated colitis-associated systemic inflammation and AKI observed in LRP5/6 LysM mice are due to increased bacterial translocation to extraintestinal sites and microbiota-dependent increased proinflammatory cytokine levels in the kidney. Conversely, depletion of the gut microbiota mitigated colitis-associated systemic inflammation and AKI in LRP5/6 LysM mice. Mechanistically, LRP5/6-deficient macrophages were hyperresponsive to TLR ligands and produced higher levels of proinflammatory cytokines, which are associated with increased activation of MAPKs. These results reveal how the Wnt-LRP5/6 signaling in macrophages controls colitis-induced systemic inflammation and AKI., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Despite recent advances in understanding the pathogenesis of polycystic kidney disease (PKD), the underlying molecular mechanisms involved in cystogenesis are not fully understood. This study describes a novel pathway involved in cyst formation. Transgenic mice overexpressing netrin-1 in proximal tubular cells showed increased production and urinary excretion of netrin-1. Although no cysts were detectable immediately after birth, numerous small cysts were evident by the age of 4 weeks, and disease was accelerated along with age. Surprisingly, cyst formation in the kidney was restricted to male mice, with 80% penetrance. However, ovariectomy induced kidney cyst growth in netrin-1-overexpressing female mice. Cyst development in males was associated with albuminuria and polyuria and increased cAMP excretion in netrin-1 transgenic mice. Netrin-1 overexpression significantly increased extracellular signal-regulated kinase and focal adhesion kinase phosphorylation and vimentin expression. Interestingly, p53 expression was increased but in an inactive form. Furthermore, netrin-1 expression was increased in cystic epithelia and urine of various rodent models of PKD. siRNA-mediated suppression of netrin-1 significantly reduced cyst growth and improved kidney function in netrin-1 transgenic mice and in two genetic animal models of PKD. Together, these data demonstrate that netrin-1 up-regulation induced cyst formation in autosomal dominant PKD., (Copyright © 2022 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Curcumin (CUR) has received great attention over the past two decades due to its anticancer, anti-inflammatory, and antioxidant properties. Similarly, Dichloroacetate (DCA), an pyruvate dehydrogenase kinase 1 (PKD1) inhibitor, has gained huge attention as a potential anticancer drug. However, the clinical utility of these two agents is very limited because of the poor bioavailability and unsolicited side effects, respectively. We have synthesized fusion conjugates of CUR and DCA with an amino acids linker to overcome these limitations by utilizing the molecular hybridization approach. The molecular docking studies showed the potential targets of Curcumin-Modified Conjugates (CMCs) in breast cancer cells. We synthesized six hybrid conjugates named CMC1-6 . These six CMC conjugates do not show any significant toxicity in a human normal immortalized mammary epithelial cell line (MCF10A) in vitro and C57BL/6 mice in vivo. However, treatment with CMC1 and CMC2 significantly reduced the growth and clonogenic survival by colony-formation assays in several human breast cancer cells (BC). Treatment by oral gavage of a transgenic mouse BC and metastatic BC tumor-bearing mice with CMC2 significantly reduced tumor growth and metastasis. Overall, our study provides strong evidence that CUR and DCA conjugates have a significant anticancer properties at a sub-micromolar concentration and overcome the clinical limitation of using CUR and DCA as potential anticancer drugs.

Metabolic reprogramming in cancer necessitates increased amino acid uptake, which is accomplished by up-regulation of specific amino acid transporters. However, not all tumors rely on any single amino acid transporter for this purpose. Here, we report on the differential up-regulation of the amino acid transporter SLC38A5 in triple-negative breast cancer (TNBC). The up-regulation is evident in TNBC tumors, conventional and patient-derived xenograft TNBC cell lines, and a mouse model of spontaneous TNBC mammary tumor. The up-regulation is confirmed by functional assays. SLC38A5 is an amino acid-dependent Na+/H+ exchanger which transports Na+ and amino acids into cells coupled with H+ efflux. Since cell-surface Na+/H+ exchanger is an established inducer of macropinocytosis, an endocytic process for cellular uptake of bulk fluid and its components, we examined the impact of SLC38A5 on macropinocytosis in TNBC cells. We found that the transport function of SLC38A5 is coupled to the induction of macropinocytosis. Surprisingly, the transport function of SLC38A5 is inhibited by amilorides, the well-known inhibitors of Na+/H+ exchanger. Down-regulation of SLC38A5 in TNBC cells attenuates serine-induced macropinocytosis and reduces cell proliferation significantly as assessed by multiple methods, but does not induce cell death. The Cancer Genome Atlas database corroborates SLC38A5 up-regulation in TNBC. This represents the first report on the selective expression of SLC38A5 in TNBC and its role as an inducer of macropinocytosis, thus revealing a novel, hitherto unsuspected, function for an amino acid transporter that goes beyond amino acid delivery but is still relevant to cancer cell nutrition and proliferation., (© 2021 The Author(s).)

Dendritic cells (DCs) are professional APCs that play a crucial role in initiating robust immune responses against invading pathogens while inducing regulatory responses to the body's tissues and commensal microorganisms. A breakdown of DC-mediated immunological tolerance leads to chronic inflammation and autoimmune disorders. However, cell-intrinsic molecular regulators that are critical for programming DCs to a regulatory state rather than to an inflammatory state are not known. In this study, we show that the activation of the TCF4 transcription factor in DCs is critical for controlling the magnitude of inflammatory responses and limiting neuroinflammation. DC-specific deletion of TCF4 in mice increased Th1/Th17 responses and exacerbated experimental autoimmune encephalomyelitis pathology. Mechanistically, loss of TCF4 in DCs led to heightened activation of p38 MAPK and increased levels of proinflammatory cytokines IL-6, IL-23, IL-1β, TNF-α, and IL-12p40. Consistent with these findings, pharmacological blocking of p38 MAPK activation delayed experimental autoimmune encephalomyelitis onset and diminished CNS pathology in TCF4 ΔDC mice. Thus, manipulation of the TCF4 pathway in DCs could provide novel opportunities for regulating chronic inflammation and represents a potential therapeutic approach to control autoimmune neuroinflammation., (Copyright © 2021 by The American Association of Immunologists, Inc.)

DNA damage, induced by either chemical carcinogens or environmental pollutants, plays an important role in the initiation of colorectal cancer. DNA repair processes, however, are involved in both protecting against cancer formation, and also contributing to cancer development, by ensuring genomic integrity and promoting the efficient DNA repair in tumor cells, respectively. Although DNA repair pathways have been well exploited in the treatment of breast and ovarian cancers, the role of DNA repair processes and their therapeutic efficacy in colorectal cancer is yet to be appreciably explored. To understand the role of DNA repair, especially homologous recombination (HR), in chemical carcinogen-induced colorectal cancer growth, we unraveled the role of RAD51AP1 (RAD51-associated protein 1), a protein involved in HR, in genotoxic carcinogen (azoxymethane, AOM)-induced colorectal cancer. Although AOM treatment alone significantly increased RAD51AP1 expression, the combination of AOM and dextran sulfate sodium (DSS) treatment dramatically increased by several folds. RAD51AP1 expression is found in mouse colonic crypt and proliferating cells. RAD51AP1 expression is significantly increased in majority of human colorectal cancer tissues, including BRAF/KRAS mutant colorectal cancer, and associated with reduced treatment response and poor prognosis. Rad51ap1 -deficient mice were protected against AOM/DSS-induced colorectal cancer. These observations were recapitulated in a genetically engineered mouse model of colorectal cancer ( Apc Min /+ ). Furthermore, chemotherapy-resistant colorectal cancer is associated with increased RAD51AP1 expression. This phenomenon is associated with reduced cell proliferation and colorectal cancer stem cell (CRCSC) self-renewal. Overall, our studies provide evidence that RAD51AP1 could be a novel diagnostic marker for colorectal cancer and a potential therapeutic target for colorectal cancer prevention and treatment. IMPLICATIONS: This study provides first in vivo evidence that RAD51AP1 plays a critical role in colorectal cancer growth and drug resistance by regulating CRCSC self-renewal., (©2021 American Association for Cancer Research.)

For decades, lactate has been considered an innocuous bystander metabolite of cellular metabolism. However, emerging studies show that lactate acts as a complex immunomodulatory molecule that controls innate and adaptive immune cells' effector functions. Thus, recent advances point to lactate as an essential and novel signaling molecule that shapes innate and adaptive immune responses in the intestine and systemic sites. Here, we review these recent advances in the context of the pleiotropic effects of lactate in regulating diverse functions of immune cells in the tissue microenvironment and under pathological conditions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Manoharan, Prasad, Thangaraju and Manicassamy.)

Oxidative damage has been identified as a major causative factor in degenerative diseases of the retina; retinal pigment epithelial (RPE) cells are at high risk. Hence, identifying novel strategies for increasing the antioxidant capacity of RPE cells, the purpose of this study, is important. Specifically, we evaluated the influence of selenium in the form of selenomethionine (Se-Met) in cultured RPE cells on system xc- expression and functional activity and on cellular levels of glutathione, a major cellular antioxidant. ARPE-19 and mouse RPE cells were cultured with and without selenomethionine (Se-Met), the principal form of selenium in the diet. Promoter activity assay, uptake assay, RT-PCR, northern and western blots, and immunofluorescence were used to analyze the expression of xc-, Nrf2, and its target genes. Se-Met activated Nrf2 and induced the expression and function of xc- in RPE. Other target genes of Nrf2 were also induced. System xc- consists of two subunits, and Se-Met induced the subunit responsible for transport activity (SLC7A11). Selenocysteine also induced xc- but with less potency. The effect of Se-met on xc- was associated with an increase in maximal velocity and an increase in substrate affinity. Se-Met increased the cellular levels of glutathione in the control, an oxidatively stressed RPE. The Se-Met effect was selective; under identical conditions, taurine transport was not affected and Na+-coupled glutamate transport was inhibited. This study demonstrates that Se-Met enhances the antioxidant capacity of RPE by inducing the transporter xc- with a consequent increase in glutathione.

Loss of immune tolerance to gut microflora is inextricably linked to chronic intestinal inflammation and colitis-associated colorectal cancer (CAC). The LRP5/6 signaling cascade in APCs contributes to immune homeostasis in the gut, but whether this pathway in APCs protects against CAC is not known. In the current study, using a mouse model of CAC, we show that the LRP5/6-β-catenin-IL-10 signaling axis in intestinal CD11c + APCs protects mice from CAC by regulating the expression of tumor-promoting inflammatory factors in response to commensal flora. Genetic deletion of LRP5/6 in CD11c + APCs in mice (LRP5/6 ΔCD11c ) resulted in enhanced susceptibility to CAC. This is due to a microbiota-dependent increased expression of proinflammatory factors and decreased expression of the immunosuppressive cytokine IL-10. This condition could be improved in LRP5/6 ΔCD11c mice by depleting the gut flora, indicating the importance of LRP5/6 in mediating immune tolerance to the gut flora. Moreover, mechanistic studies show that LRP5/6 suppresses the expression of tumor-promoting inflammatory factors in CD11c + APCs via the β-catenin-IL-10 axis. Accordingly, conditional activation of β-catenin specifically in CD11c + APCs or in vivo administration of IL-10 protected LRP5/6 ΔCD11c mice from CAC by suppressing the expression of inflammatory factors. In summary, in this study, we identify a key role for the LRP5/6-β-catenin-IL-10 signaling pathway in intestinal APCs in resolving chronic intestinal inflammation and protecting against CAC in response to the commensal flora., (Copyright © 2020 by The American Association of Immunologists, Inc.)

RAD51-associated protein 1 (RAD51AP1) plays an integral role in homologous recombination by activating RAD51 recombinase. Homologous recombination is essential for preserving genome integrity and RAD51AP1 is critical for D-loop formation, a key step in homologous recombination. Although RAD51AP1 is involved in maintaining genomic stability, recent studies have shown that RAD51AP1 expression is significantly upregulated in human cancers. However, the functional role of RAD51AP1 in tumor growth and the underlying molecular mechanism(s) by which RAD51AP1 regulates tumorigenesis have not been fully understood. Here, we use Rad51ap1-knockout mice in genetically engineered mouse models of breast cancer to unravel the role of RAD51AP1 in tumor growth and metastasis. RAD51AP1 gene transcript was increased in both luminal estrogen receptor-positive breast cancer and basal triple-negative breast cancer, which is associated with poor prognosis. Conversely, knockdown of RAD51AP1 (RADP51AP1 KD) in breast cancer cell lines reduced tumor growth. Rad51ap1-deficient mice were protected from oncogene-driven spontaneous mouse mammary tumor growth and associated lung metastasis. In vivo , limiting dilution studies provided evidence that Rad51ap1 plays a critical role in breast cancer stem cell (BCSC) self-renewal. RAD51AP1 KD improved chemotherapy and radiotherapy response by inhibiting BCSC self-renewal and associated pluripotency. Overall, our study provides genetic and biochemical evidences that RAD51AP1 is critical for tumor growth and metastasis by increasing BCSC self-renewal and may serve as a novel target for chemotherapy- and radiotherapy-resistant breast cancer. SIGNIFICANCE: This study provides in vivo evidence that RAD51AP1 plays a critical role in breast cancer growth and metastasis by regulating breast cancer stem cell self-renewal., (©2020 American Association for Cancer Research.)

The 5-year survival rate of patients with metastatic renal cell carcinoma (mRCC) is <12% due to treatment failure. Therapeutic strategies that overcome resistance to modestly effective drugs for mRCC, such as sorafenib (SF), could improve outcome in mRCC patients. SF is terminally biotransformed by UDP-glucuronosyltransferase-1A9 (A9) mediated glucuronidation, which inactivates SF. In a clinical-cohort and the TCGA-dataset, A9 transcript and/or protein levels were highly elevated in RCC specimens and predicted metastasis and overall-survival. This suggested that elevated A9 levels even in primary tumors of patients who eventually develop mRCC could be a mechanism for SF failure. 4-methylumbelliferone (MU), a choleretic and antispasmodic drug, downregulated A9 and inhibited SF-glucuronidation in RCC cells. Low-dose SF and MU combinations inhibited growth, motility, invasion and downregulated an invasive signature in RCC cells, patient-derived tumor explants and/or endothelial-RCC cell co-cultures; however, both agents individually were ineffective. A9 overexpression made RCC cells resistant to the combination, while its downregulation sensitized them to SF treatment alone. The combination inhibited kidney tumor growth, angiogenesis and distant metastasis, with no detectable toxicity; A9-overexpressing tumors were resistant to treatment. With effective primary tumor control and abrogation of metastasis in preclinical models, the low-dose SF and MU combinations could be an effective treatment option for mRCC patients. Broadly, our study highlights how targeting specific mechanisms that cause the failure of "old" modestly effective FDA-approved drugs could improve treatment response with minimal alteration in toxicity profile.

SLC6A14 is a Na+/Cl--coupled transporter for neutral and cationic amino acids. It is expressed at basal levels in the normal colon but is up-regulated in colon cancer. However, the relevance of this up-regulation to cancer progression and the mechanisms involved in the up-regulation remain unknown. Here, we show that SLC6A14 is essential for colon cancer and that its up-regulation involves, at least partly, Wnt signaling. The up-regulation of the transporter is evident in most human colon cancer cell lines and also in a majority of patient-derived xenografts. These findings are supported by publicly available TCGA (The Cancer Genome Atlas) database. Treatment of colon cancer cells with α-methyltryptophan (α-MT), a blocker of SLC6A14, induces amino acid deprivation, decreases mTOR activity, increases autophagy, promotes apoptosis, and suppresses cell proliferation and invasion. In xenograft and syngeneic mouse tumor models, silencing of SLC6A14 by shRNA or blocking its function by α-MT reduces tumor growth. Similarly, the deletion of Slc6a14 in mice protects against colon cancer in two different experimental models (inflammation-associated colon cancer and genetically driven colon cancer). In colon cancer cells, expression of the transporter is reduced by Wnt antagonist or by silencing of β-catenin whereas Wnt agonist or overexpression of β-catenin shows the opposite effect. Finally, SLC6A14 as a target for β-catenin is confirmed by chromatin immunoprecipitation. These studies demonstrate that SLC6A14 plays a critical role in the promotion of colon cancer and that its up-regulation in cancer involves Wnt signaling. These findings identify SLC6A14 as a promising drug target for the treatment of colon cancer., (© 2020 The Author(s).)

Based on the evidence that hemochromatosis, an iron-overload disease, drives hepatocellular carcinoma, we hypothesized that chronic exposure to excess iron, either due to genetic or environmental causes, predisposes an individual to cancer. Using pancreatic cancer as our primary focus, we employed cell culture studies to interrogate the connection between excess iron and cancer, and combined in vitro and in vivo studies to explore the connection further. Ferric ammonium citrate was used as an exogenous iron source. Chronic exposure to excess iron induced epithelial-mesenchymal transition (EMT) in normal and cancer cell lines, loss of p53, and suppression of p53 transcriptional activity evidenced from decreased expression of p53 target genes (p21, cyclin D1, Bax, SLC7A11). To further extrapolate our cell culture data, we generated EL - Kras G12D ( EL-Kras ) mouse (pancreatic neoplastic mouse model) expressing Hfe +/+ and Hfe -/- genetic background. p53 target gene expression decreased in EL-Kras / Hfe -/- mouse pancreas compared to EL-Kras / Hfe +/+ mouse pancreas. Interestingly, the incidence of acinar-to-ductal metaplasia and cystic pancreatic neoplasms (CPN) decreased in EL-Kras / Hfe -/- mice, but the CPNs that did develop were larger in these mice than in EL-Kras / Hfe +/+ mice. In conclusion, these in vitro and in vivo studies support a potential role for chronic exposure to excess iron as a promoter of more aggressive disease via p53 loss and SLC7A11 upregulation within pancreatic epithelial cells., Competing Interests: The authors declare that they have no potential conflicts of interest., (© 2020 Shenyang Pharmaceutical University. Published by Elsevier B.V.)

The WASF3 gene has been implicated in cancer cell movement, invasion, and metastasis by regulating genetic pathways important in these processes. Invasion and metastasis assays, however, are largely centered on xenograft models in immune-compromised mice. To facilitate analysis of the role of WASF3 in the spontaneous development of cancer cell metastasis, we generated a Wasf3 null strain by deleting exons 4 and 5, which encode essential motifs for Wasf3 function. On exposure to cre-recombinase a stop codon is generated immediately downstream in exon 6. Using a cytomegalovirus (CMV)-cre strain, Wasf3 constitutively was inactivated, which led to viable mice with no visible morphologic or behavioral abnormalities. There was no abnormal development or function of the mouse mammary gland in the Wasf3 null mice and brain development was normal. In the mouse mammary tumor virus (MMTV)-driven polyoma middle-T oncogene strain, which shows early onset breast cancer development and metastasis, Wiskott-Aldrich syndrome protein family member 3 (Wasf3) is up-regulated in metastatic lesions. When this oncogene was introduced onto the Wasf3-null background, although metastasis was observed in these mice, there was a reduction in the number and size of metastatic lesions in the lungs. These data provide evidence for a role in WASF3 in the development of metastasis in a spontaneous model of breast cancer., (Copyright © 2019 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

MYCN amplification drives the development of neuronal cancers in children and adults. Given the challenge in therapeutically targeting MYCN directly, we searched for MYCN-activated metabolic pathways as potential drug targets. Here we report that neuroblastoma cells with MYCN amplification show increased transcriptional activation of the serine-glycine-one-carbon (SGOC) biosynthetic pathway and an increased dependence on this pathway for supplying glucose-derived carbon for serine and glycine synthesis. Small molecule inhibitors that block this metabolic pathway exhibit selective cytotoxicity to MYCN -amplified cell lines and xenografts by inducing metabolic stress and autophagy. Transcriptional activation of the SGOC pathway in MYCN -amplified cells requires both MYCN and ATF4, which form a positive feedback loop, with MYCN activation of ATF4 mRNA expression and ATF4 stabilization of MYCN protein by antagonizing FBXW7-mediated MYCN ubiquitination. Collectively, these findings suggest a coupled relationship between metabolic reprogramming and increased sensitivity to metabolic stress, which could be exploited as a strategy for selective cancer therapy. SIGNIFICANCE: This study identifies a MYCN-dependent metabolic vulnerability and suggests a coupled relationship between metabolic reprogramming and increased sensitivity to metabolic stress, which could be exploited for cancer therapy. See related commentary by Rodriguez Garcia and Arsenian-Henriksson, p. 3818 ., (©2019 American Association for Cancer Research.)

Although clinically apparent metastasis is associated with late stages of cancer development, micro-metastatic dissemination may be an early event. However, the fate of these early disseminated tumor cells (DTC) remains elusive. We show that despite their capacity to disseminate into secondary organs, 4T1 tumor models develop overt metastasis while EMT6-tumor bearing mice clear DTCs shed from primary tumors as well as those introduced by intravenous (IV) injection. Following the surgical resection of primary EMT6 tumors, mice do not develop detectable metastasis and reject IV-injected tumor cells. In contrast, these cells readily grow and metastasize in immuno-deficient athymic or Rag2 -/- mice, an effect mimicked by CD8 + T-cell depletion in immunocompetent mice. Furthermore, recombinant G-CSF or adoptive transfer of granulocytic-MDSCs isolated from 4T1 tumor-bearing mice, induce metastasis by suppressing CD8 + T-cells in EMT6-primed mice. Our studies support the concept of immune surveillance providing molecular insights into the immune mechanisms during tumor progression.

Deferiprone (DFP) is a hydroxypyridinone-derived iron chelator currently in clinical use for iron chelation therapy. DFP has also been known to elicit antiproliferative activities, yet the mechanism of this effect has remained elusive. We herein report that DFP chelates the Fe 2+ ion at the active sites of selected iron-dependent histone lysine demethylases (KDMs), resulting in pan inhibition of a subfamily of KDMs. Specifically, DFP inhibits the demethylase activities of six KDMs - 2A, 2B, 5C, 6A, 7A and 7B - with low micromolar IC 50 s while considerably less active or inactive against eleven KDMs - 1A, 3A, 3B, 4A-E, 5A, 5B and 6B. The KDM that is most sensitive to DFP, KDM6A, has an IC 50 that is between 7- and 70-fold lower than the iron binding equivalence concentrations at which DFP inhibits ribonucleotide reductase (RNR) activities and/or reduces the labile intracellular zinc ion pool. In breast cancer cell lines, DFP potently inhibits the demethylation of H3K4me3 and H3K27me3, two chromatin posttranslational marks that are subject to removal by several KDM subfamilies which are inhibited by DFP in cell-free assay. These data strongly suggest that DFP derives its anti-proliferative activity largely from the inhibition of a sub-set of KDMs. The docked poses adopted by DFP at the KDM active sites enabled identification of new DFP-based KDM inhibitors which are more cytotoxic to cancer cell lines. We also found that a cohort of these agents inhibited HP1-mediated gene silencing and one lead compound potently inhibited breast tumor growth in murine xenograft models. Overall, this study identified a new chemical scaffold capable of inhibiting KDM enzymes, globally changing histone modification profiles, and with specific anti-tumor activities.

Alcoholic ketoacidosis and diabetic ketoacidosis are life-threatening complications that share the characteristic features of high anion gap metabolic acidosis. Ketoacidosis is attributed in part to the massive release of ketone bodies (e.g., β-hydroxybutyrate; βOHB) from the liver into the systemic circulation. To date, the impact of ethanol consumption on systemic ketone concentration, glycemic control, and hepatic gluconeogenesis and glycogenesis remains largely unknown, especially in the context of type 2 diabetes. In the present study, ethanol intake (36% ethanol- and 36% fat-derived calories) by type 2 diabetic db/db mice for 9 days resulted in significant decreases in weight gain (∼19.5% ↓) and caloric intake (∼30% ↓). This was accompanied by a transition from macrovesicular-to-microvesicular hepatic steatosis with a modest increase in hepatic TG (∼37% ↑). Importantly, ethanol increased systemic βOHB concentration (∼8-fold ↑) with significant decreases in blood glucose (∼4-fold ↓) and plasma insulin and HOMA-IR index (∼3-fold ↓). In addition, ethanol enhanced hepatic βOHB content (∼5-fold ↑) and hmgcs2 mRNA expression (∼3.7-fold ↑), downregulated key gluconeogenic mRNAs (e.g., Pcx, Pck1, and G6pc), and depleted hepatic glycogen (∼4-fold ↓). Furthermore, ethanol intake led to significant decreases in the mRNA/protein expression and allosteric activation of glycogen synthase (GS) in liver tissues regardless of changes in the phosphorylation of GS, GSK-3β, or Akt. Together, our findings suggest that ethanol-induced ketonemia may occur in concomitance with significant lowering of blood glucose concentration, which may be attributed to suppression of gluconeogenesis in the setting of glycogen depletion in type 2 diabetes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Aims: Invasion and metastasis are the main cause of mortality in breast cancer. Hence, novel therapeutic interventions with high specificity toward invasion and metastasis are necessary. α-Lipoic acid showed antiproliferative and cytotoxic effects on several cancers including breast cancer. However, the effect of lipoic acid on breast cancer metastasis remains unclear., Main Methods: In the present study, we examined the effects of lipoic acid on the migration and invasion of MDA-MB-231 and 4T1 breast cancer cells., Key Findings: Our data showed that lipoic acid effectively inhibited the colony forming ability of highly invasive MDA-MB-231 and 4T1 cells. Moreover, the nontoxic concentrations of lipoic acid significantly reduced the migration of breast cancer cells. Lipoic acid also inhibited the TGFβ-induced angiopoietin-like 4 (ANGPTL4) expression and reduced the activity of matrix metalloproteinase-9 (MMP-9), an enzyme involved in invasion and metastasis, in both the cell lines. The inhibition of cell migration by lipoic acid is accompanied by the downregulation of FAK, ERK1/2 and AKT phosphorylation, and inhibition of nuclear translocation of β-catenin., Significance: Our data demonstrated that lipoic acid inhibited the migration and invasion of metastatic breast cancer cells at least in part through inhibiting ERK1/2 and AKT signaling. Thus, our findings show that the inhibition of TGFβ signaling is a potential mechanism for the anti-invasive effects of lipoic acid., (Copyright © 2018. Published by Elsevier Inc.)

Aberrant Wnt/β-catenin signaling occurs in several inflammatory diseases, including inflammatory bowel disease and inflammatory bowel disease-associated colon carcinogenesis. However, its role in shaping mucosal immune responses to commensals in the gut remains unknown. In this study, we investigated the importance of canonical Wnt signaling in CD11c + APCs in controlling intestinal inflammation. Using a mouse model of ulcerative colitis, we demonstrated that canonical Wnt signaling in intestinal CD11c + APCs controls intestinal inflammation by imparting an anti-inflammatory phenotype. Genetic deletion of Wnt coreceptors, low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) in CD11c + APCs in LRP5/6 ΔCD11c mice, resulted in enhanced intestinal inflammation with increased histopathological severity of colonic tissue. This was due to microbiota-dependent increased production of proinflammatory cytokines and decreased expression of immune-regulatory factors such as IL-10, retinoic acid, and IDO. Mechanistically, loss of LRP5/6-mediated signaling in CD11c + APCs resulted in altered microflora and T cell homeostasis. Furthermore, our study demonstrates that conditional activation of β-catenin in CD11c + APCs in LRP5/6 ΔCD11c mice resulted in reduced intestinal inflammation with decreased histopathological severity of colonic tissue. These results reveal a mechanism by which intestinal APCs control intestinal inflammation and immune homeostasis via the canonical Wnt-signaling pathway., (Copyright © 2018 by The American Association of Immunologists, Inc.)

At mucosal sites such as the intestine, the immune system launches robust immunity against invading pathogens while maintaining a state of tolerance to commensal flora and ingested food Ags. The molecular mechanisms underlying this phenomenon remain poorly understood. In this study, we report that signaling by GPR81, a receptor for lactate, in colonic dendritic cells and macrophages plays an important role in suppressing colonic inflammation and restoring colonic homeostasis. Genetic deletion of GPR81 in mice led to increased Th1/Th17 cell differentiation and reduced regulatory T cell differentiation, resulting in enhanced susceptibility to colonic inflammation. This was due to increased production of proinflammatory cytokines (IL-6, IL-1β, and TNF-α) and decreased expression of immune regulatory factors (IL-10, retinoic acid, and IDO) by intestinal APCs lacking GPR81. Consistent with these findings, pharmacological activation of GPR81 decreased inflammatory cytokine expression and ameliorated colonic inflammation. Taken together, these findings identify a new and important role for the GPR81 signaling pathway in regulating immune tolerance and colonic inflammation. Thus, manipulation of the GPR81 pathway could provide novel opportunities for enhancing regulatory responses and treating colonic inflammation., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Diabetic retinopathy (DR) is a leading cause of blindness among working-age adults. Increased iron accumulation is associated with several degenerative diseases. However, there are no reports on the status of retinal iron or its implications in the pathogenesis of DR. In the present study, we found that retinas of type-1 and type-2 mouse models of diabetes have increased iron accumulation compared to non-diabetic retinas. We found similar iron accumulation in postmortem retinal samples from human diabetic patients. Further, we induced diabetes in HFE knockout (KO) mice model of genetic iron overload to understand the role of iron in the pathogenesis of DR. We found increased neuronal cell death, vascular alterations and loss of retinal barrier integrity in diabetic HFE KO mice compared to diabetic wildtype mice. Diabetic HFE KO mouse retinas also exhibited increased expression of inflammation and oxidative stress markers. Severity in the pathogenesis of DR in HFE KO mice was accompanied by increase in retinal renin expression mediated by G-protein-coupled succinate receptor GPR91. In light of previous reports implicating retinal renin-angiotensin system in DR pathogenesis, our results reveal a novel relationship between diabetes, iron and renin-angiotensin system, thereby unraveling new therapeutic targets for the treatment of DR.

It is widely accepted that dynamic and reversible tumour cell plasticity is required for metastasis, however, in vivo steps and molecular mechanisms are poorly elucidated. We demonstrate here that monocytic (mMDSC) and granulocytic (gMDSC) subsets of myeloid-derived suppressor cells infiltrate in the primary tumour and distant organs with different time kinetics and regulate spatiotemporal tumour plasticity. Using co-culture experiments and mouse transcriptome analyses in syngeneic mouse models, we provide evidence that tumour-infiltrated mMDSCs facilitate tumour cell dissemination from the primary site by inducing EMT/CSC phenotype. In contrast, pulmonary gMDSC infiltrates support the metastatic growth by reverting EMT/CSC phenotype and promoting tumour cell proliferation. Furthermore, lung-derived gMDSCs isolated from tumour-bearing animals enhance metastatic growth of already disseminated tumour cells. MDSC-induced 'metastatic gene signature' derived from murine syngeneic model predicts poor patient survival in the majority of human solid tumours. Thus spatiotemporal MDSC infiltration may have clinical implications in tumour progression.

Triple-negative breast cancer (TNBC) cells are modulated in reaction to tumor-infiltrating lymphocytes. However, their specific responses to this immune pressure are unknown. In order to address this question, we first used mRNA sequencing to compare the immunophenotype of the TNBC cell line MDA-MB-231 and the luminal breast cancer cell line MCF7 after both were cocultured with activated human T cells. Despite similarities in the cytokine-induced immune signatures of the two cell lines, MDA-MD-231 cells were able to transcribe more IDO1 than MCF7 cells. The two cell lines had similar upstream JAK/STAT1 signaling and IDO1 mRNA stability. However, using a series of breast cancer cell lines, IFNγ stimulated IDO1 protein expression and enzymatic activity only in ER - , not ER + , cell lines. Treatment with 5-aza-deoxycytidine reversed the suppression of IDO1 expression in MCF7 cells, suggesting that DNA methylation was potentially involved in IDO1 induction. By analyzing several breast cancer datasets, we discovered subtype-specific mRNA and promoter methylation differences in IDO1 , with TNBC/basal subtypes exhibiting lower methylation/higher expression and ER + /luminal subtypes exhibiting higher methylation/lower expression. We confirmed this trend of IDO1 methylation by bisulfite pyrosequencing breast cancer cell lines and an independent cohort of primary breast tumors. Taken together, these findings suggest that IDO1 promoter methylation regulates anti-immune responses in breast cancer subtypes and could be used as a predictive biomarker for IDO1 inhibitor-based immunotherapy. Cancer Immunol Res; 5(4); 330-44. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Dietary lipids and their metabolites activate members of the peroxisome proliferative-activated receptor (PPAR) family of transcription factors and are critical for colonic health. The PPARα isoform plays a vital role in regulating inflammation in various disease settings, but its role in intestinal inflammation, commensal homeostasis, and mucosal immunity in the gut are unclear. In this study, we demonstrate that the PPARα pathway in innate immune cells orchestrates gut mucosal immunity and commensal homeostasis by regulating the expression of IL-22 and the antimicrobial peptides RegIIIβ, RegIIIγ, and calprotectin. Additionally, the PPARα pathway is critical for imparting regulatory phenotype in intestinal macrophages. PPARα deficiency in mice led to commensal dysbiosis in the gut, resulting in a microbiota-dependent increase in the expression of inflammatory cytokines and enhanced susceptibility to intestinal inflammation. Pharmacological activation of this pathway decreased the expression of inflammatory cytokines and ameliorated colonic inflammation. Taken together, these findings identify a new important innate immune function for the PPARα signaling pathway in regulating intestinal inflammation, mucosal immunity, and commensal homeostasis. Thus, the manipulation of the PPARα pathway could provide novel opportunities for enhancing mucosal immunity and treating intestinal inflammation., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Recently, impressive technical advancements have been made in the isolation and validation of mammary stem cells and cancer stem cells (CSC), but the signaling pathways that regulate stem cell self-renewal are largely unknown. Furthermore, CSCs are believed to contribute to chemo- and radioresistance. In this study, we used the MMTV-Neu-Tg mouse mammary tumor model to identify potential new strategies for eliminating CSCs. We found that both luminal progenitor and basal stem cells are susceptible to genetic and epigenetic modifications, which facilitate oncogenic transformation and tumorigenic potential. A combination of the DNMT inhibitor 5-azacytidine and the HDAC inhibitor butyrate markedly reduced CSC abundance and increased the overall survival in this mouse model. RNA-seq analysis of CSCs treated with 5-azacytidine plus butyrate provided evidence that inhibition of chromatin modifiers blocks growth-promoting signaling molecules such as RAD51AP1 and SPC25, which play key roles in DNA damage repair and kinetochore assembly. Moreover, RAD51AP1 and SPC25 were significantly overexpressed in human breast tumor tissues and were associated with reduced overall patient survival. In conclusion, our studies suggest that breast CSCs are intrinsically sensitive to genetic and epigenetic modifications and can therefore be significantly affected by epigenetic-based therapies, warranting further investigation of combined DNMT and HDAC inhibition in refractory or drug-resistant breast cancer. Cancer Res; 76(11); 3224-35. ©2016 AACR., (©2016 American Association for Cancer Research.)

The role of plasma membrane transporters in cancer is receiving increasing attention in recent years. Several transporters for essential nutrients are up-regulated in cancer and serve as tumour promoters. Transporters could also function as tumour suppressors. To date, four transporters belonging to the SLC gene family have been identified as tumour suppressors. SLC5A8 is a Na(+)-coupled transporter for monocarboxylates. Among its substrates are the bacterial fermentation products butyrate and propionate and the ubiquitous metabolite pyruvate. The tumour-suppressive function of this transporter relates to the ability of butyrate, propionate and pyruvate to inhibit histone deacetylases (HDAC). SLC5A8 functions as a tumour suppressor in most tissues studied thus far, and provides a molecular link to Warburg effect, a characteristic feature in most cancers. It also links colonic bacteria and dietary fibre to the host. SLC26A3 as a tumour suppressor is restricted to colon; it is a Cl(-)/HCO(-) 3 exchanger, facilitating the efflux of HCO(-) 3 The likely mechanism for the tumour-suppressive function of SLC26A3 is related to intracellular pH regulation. SLC39A1 is a Zn(2+) transporter and its role in tumour suppression has been shown in prostate. Zn(2+) is present at high concentrations in normal prostate where it elicits its tumour-suppressive function. SLC22A18 is possibly an organic cation transporter, but the identity of its physiological substrates is unknown. As such, there is no information on molecular pathways responsible for the tumour-suppressive function of this transporter. It is likely that additional SLC transporters will be discovered as tumour suppressors in the future., (© 2016 Authors; published by Portland Press Limited.)