Intestinal Bacteroides modulates inflammation, systemic cytokines, and microbial ecology via propionate in a mouse model of cystic fibrosis.

Persons with cystic fibrosis (CF), starting in early life, show intestinal microbiome dysbiosis characterized in part by a decreased relative abundance of the genus Bacteroides. Bacteroides is a major producer of the intestinal short chain fatty acid propionate. We demonstrate here that cystic fibrosis transmembrane conductance regulator-defective (CFTR-/-) Caco-2 intestinal epithelial cells are responsive to the anti-inflammatory effects of propionate. Furthermore, Bacteroides isolates inhibit the IL-1ß-induced inflammatory response of CFTR-/- Caco-2 intestinal epithelial cells and do so in a propionate-dependent manner. The introduction of Bacteroides-supplemented stool from infants with cystic fibrosis into the gut of CftrF508del mice results in higher propionate in the stool as well as the reduction in several systemic pro-inflammatory cytokines. Bacteroides supplementation also reduced the fecal relative abundance of Escherichia coli, indicating a potential interaction between these two microbes, consistent with previous clinical studies. For a Bacteroides propionate mutant in the mouse model, pro-inflammatory cytokine KC is higher in the airway and serum compared with the wild-type (WT) strain, with no significant difference in the absolute abundance of these two strains. Taken together, our data indicate the potential multiple roles of Bacteroides-derived propionate in the modulation of systemic and airway inflammation and mediating the intestinal ecology of infants and children with CF. The roles of Bacteroides and the propionate it produces may help explain the observed gut-lung axis in CF and could guide the development of probiotics to mitigate systemic and airway inflammation for persons with CF.IMPORTANCEThe composition of the gut microbiome in persons with CF is correlated with lung health outcomes, a phenomenon referred to as the gut-lung axis. Here, we demonstrate that the intestinal microbe Bacteroides decreases inflammation through the production of the short-chain fatty acid propionate. Supplementing the levels of Bacteroides in an animal model of CF is associated with reduced systemic inflammation and reduction in the relative abundance of the opportunistically pathogenic group Escherichia/Shigella in the gut. Taken together, these data demonstrate a key role for Bacteroides and microbially produced propionate in modulating inflammation, gut microbial ecology, and the gut-lung axis in cystic fibrosis. These data support the role of Bacteroides as a potential probiotic in CF.

Community composition and the environment modulate the population dynamics of type VI secretion in human gut bacteria.

Understanding the relationship between the composition of the human gut microbiota and the ecological forces shaping it is of great importance; however, knowledge of the biogeographical and ecological relationships between physically interacting taxa is limited. Interbacterial antagonism may play an important role in gut community dynamics, yet the conditions under which antagonistic behaviour is favoured or disfavoured by selection in the gut are not well understood. Here, using genomics, we show that a species-specific type VI secretion system (T6SS) repeatedly acquires inactivating mutations in Bacteroides fragilis in the human gut. This result implies a fitness cost to the T6SS, but we could not identify laboratory conditions under which such a cost manifests. Strikingly, experiments in mice illustrate that the T6SS can be favoured or disfavoured in the gut depending on the strains and species in the surrounding community and their susceptibility to T6SS antagonism. We use ecological modelling to explore the conditions that could underlie these results and find that community spatial structure modulates interaction patterns among bacteria, thereby modulating the costs and benefits of T6SS activity. Our findings point towards new integrative models for interrogating the evolutionary dynamics of type VI secretion and other modes of antagonistic interaction in microbiomes.

Community composition and the environment modulate the population dynamics of type VI secretion in human gut bacteria.

Understanding the relationship between the composition of the human gut microbiota and the ecological forces shaping it is of high importance as progress towards therapeutic modulation of the microbiota advances. However, given the inaccessibility of the gastrointestinal tract, our knowledge of the biogeographical and ecological relationships between physically interacting taxa has been limited to date. It has been suggested that interbacterial antagonism plays an important role in gut community dynamics, but in practice the conditions under which antagonistic behavior is favored or disfavored by selection in the gut environment are not well known. Here, using phylogenomics of bacterial isolate genomes and analysis of infant and adult fecal metagenomes, we show that the contact-dependent type VI secretion system (T6SS) is repeatedly lost from the genomes of Bacteroides fragilis in adults compare to infants. Although this result implies a significant fitness cost to the T6SS, but we could not identify in vitro conditions under which such a cost manifests. Strikingly, however, experiments in mice illustrated that the B. fragilis T6SS can be favored or disfavored in the gut environment, depending on the strains and species in the surrounding community and their susceptibility to T6SS antagonism. We use a variety of ecological modeling techniques to explore the possible local community structuring conditions that could underlie the results of our larger scale phylogenomic and mouse gut experimental approaches. The models illustrate robustly that the pattern of local community structuring in space can modulate the extent of interactions between T6SS-producing, sensitive, and resistant bacteria, which in turn control the balance of fitness costs and benefits of performing contact-dependent antagonistic behavior. Taken together, our genomic analyses, in vivo studies, and ecological theory point toward new integrative models for interrogating the evolutionary dynamics of type VI secretion and other predominant modes of antagonistic interaction in diverse microbiomes.

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice.

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the CSF protein composition delivers crucial information in basic neuroscience research as well as neurological diseases. One caveat is that proteins measured in CSF may derive from both intrathecal synthesis and transudation from serum, and protein analysis of CSF can only determine the sum of these two components. To discriminate between protein transudation from the blood and intrathecally produced proteins in animal models as well as in humans, CSF protein profiling measurements using conventional protein analysis tools must include the calculation of the albumin CSF/serum quotient (Qalbumin), a marker of the integrity of the blood-brain interface (BBI), and the protein index (Qprotein/Qalbumin), an estimate of intrathecal protein synthesis. This protocol illustrates the entire procedure, from CSF and blood collection to quotients and indices calculations, for the quantitative measurement of intrathecal protein synthesis and BBI impairment in mouse models of neurological disorders.

Central Nervous System Inflammatory Aggregates in the Theiler's Virus Model of Progressive Multiple Sclerosis.

Persistent central nervous system (CNS) inflammation, as seen in chronic infections or inflammatory demyelinating diseases such as Multiple Sclerosis (MS), results in the accumulation of various B cell subsets in the CNS, including naïve, activated, memory B cells (Bmem), and antibody secreting cells (ASC). However, factors driving heterogeneous B cell subset accumulation and antibody (Ab) production in the CNS compartment, including the contribution of ectopic lymphoid follicles (ELF), during chronic CNS inflammation remain unclear and is a major gap in our understanding of neuroinflammation. We sought to address this gap using the Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) model of progressive MS. In this model, injection of the virus into susceptible mouse strains results in a persistent infection associated with demyelination and progressive disability. During chronic infection, the predominant B cell phenotypes accumulating in the CNS were isotype-switched B cells, including Bmem and ASC with naïve/early activated and transitional B cells present at low frequencies. B cell accumulation in the CNS during chronic TMEV-IDD coincided with intrathecal Ab synthesis in the cerebrospinal fluid (CSF). Mature and isotype-switched B cells predominately localized to the meninges and perivascular space, with IgG isotype-switched B cells frequently accumulating in the parenchymal space. Both mature and isotype-switched B cells and T cells occupied meningeal and perivascular spaces, with minimal evidence for spatial organization typical of ELF mimicking secondary lymphoid organs (SLO). Moreover, immunohistological analysis of immune cell aggregates revealed a lack of SLO-like ELF features, such as cell proliferation, cell death, and germinal center B cell markers. Nonetheless, flow cytometric assessment of B cells within the CNS showed enhanced expression of activation markers, including moderate upregulation of GL7 and expression of the costimulatory molecule CD80. B cell-related chemokines and trophic factors, including APRIL, BAFF, CXCL9, CXCL10, CCL19, and CXCL13, were elevated in the CNS. These results indicate that localization of heterogeneous B cell populations, including activated and isotype-switched B cell phenotypes, to the CNS and intrathecal Ab (ItAb) synthesis can occur independently of SLO-like follicles during chronic inflammatory demyelinating disease.

Differential neuro-immune patterns in two clinically relevant murine models of multiple sclerosis.

BACKGROUND: The mechanisms driving multiple sclerosis (MS), the most common cause of non-traumatic disability in young adults, remain unknown despite extensive research. Especially puzzling are the underlying molecular processes behind the two major disease patterns of MS: relapsing-remitting and progressive. The relapsing-remitting course is exemplified by acute inflammatory attacks, whereas progressive MS is characterized by neurodegeneration on a background of mild-moderate inflammation. The molecular and cellular features differentiating the two patterns are still unclear, and the role of inflammation during progressive disease is a subject of active debate. METHODS: We performed a comprehensive analysis of the intrathecal inflammation in two clinically distinct mouse models of MS: the PLP139-151-induced relapsing experimental autoimmune encephalomyelitis (R-EAE) and the chronic progressive, Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD). Microarray technology was first used to examine global gene expression changes in the spinal cord. Inflammation in the spinal cord was further assessed by immunohistochemical image analysis and flow cytometry. Levels of serum and cerebrospinal fluid (CSF) immunoglobulin (Ig) isotypes and chemokines were quantitated using Luminex Multiplex technology, whereas a capture ELISA was used to measure serum and CSF albumin levels. Finally, an intrathecal Ig synthesis index was established with the ratio of CSF and serum test results corrected as a ratio of their albumin concentrations. RESULTS: Microarray analysis identified an enrichment of B cell- and Ig-related genes upregulated in TMEV-IDD mice. We also demonstrated an increased level of intrathecal Ig synthesis as well as a marked infiltration of late differentiated B cells, including antibody secreting cells (ASC), in the spinal cord of TMEV-IDD, but not R-EAE mice. An intact blood-brain barrier in TMEV-IDD mice along with higher CSF levels of CXCL13, CXCL12, and CCL19 provides evidence for an intrathecal synthesis of chemokines mediating B cell localization to the central nervous system (CNS). CONCLUSIONS: Overall, these findings, showing increased concentrations of intrathecally produced Igs, substantial infiltration of ASC, and the presence of B cell supporting chemokines in the CNS of TMEV-IDD mice, but not R-EAE mice, suggest a potentially important role for Igs and ASC in the chronic progressive phase of demyelinating diseases.

Clinical utility of a molecular signature in inflammatory demyelinating disease.

Objective: We sought to develop molecular biomarkers of intrathecal inflammation to assist neurologists in identifying patients most likely to benefit from a range of immune therapies. Methods: We used Luminex technology and index determination to search for an inflammatory activity molecular signature (IAMS) in patients with inflammatory demyelinating disease (IDD), other neuroinflammatory diagnoses, and noninflammatory controls. We then followed the clinical characteristics of these patients to find how the presence of the signature might assist in diagnosis and prognosis. Results: A CSF molecular signature consisting of elevated CXCL13, elevated immunoglobulins, normal albumin CSF/serum ratio (Qalbumin), and minimal elevation of cytokines other than CXCL13 provided diagnostic and prognostic value; absence of the signature in IDD predicted lack of subsequent inflammatory events. The signature outperformed oligoclonal bands, which were frequently false positive for active neuroinflammation. Conclusions: A CSF IAMS may prove useful in the diagnosis and management of patients with IDD and other neuroinflammatory syndromes. Classification of evidence: This study provides Class IV evidence that a CSF IAMS identifies patients with IDD.

Interaction of PEGylated interferon-beta with antibodies to recombinant interferon-beta.

Because PEGylated molecules exhibit different physicochemical properties from those of the parent molecules, PEGylated interferonß-1a (pegIFNß-1a) may be able to be used with retained bioactivity in Multiple Sclerosis (MS) patients who have previously developed neutralizing antibodies (NABs) to recombinant interferonß (rIFNß). Hence, the objective of the present study was to test whether pegIFNß-1a is less antigenic for NABs in vitro than rIFNß. Two in vitro assays were used to quantitate NABs in 115 sera obtained from MS patients included in the INSIGHT study: the cytopathic effect (CPE) assay, and the MxA protein induction assay. NABs cross-reactivity was assessed by comparing dilutions of serum with fixed doses of rIFNß-1a Avonex® and pegIFNß-1a Plegridy®. NABs were shown to cross-react in both assays. The y-intercept (c), the slope of the line of agreement (b), the Pearson coefficients as well as the Bland-Altman analysis, indicated that there is good level of agreement between NAB titers against the two IFNß-1a formulations, with both the CPE (c = 0.1044 ±â€¯0.1305; b = 0.8438 ±â€¯0.06654; r2 = 0.587; bias index ±â€¯SD = -0.01702 ±â€¯0.6334), and the MxA protein induction (c = 0.08246 ±â€¯0.1229; b = 0.8878 ±â€¯0.06613; r2 = 0.615; bias index ±â€¯SD = -0.09965 ±â€¯0.6467) assays. Until further in vivo evidence is established, clinicians should consider the current in vitro data demonstrating NAB cross-reactivity between pegIFNß-1a and rIFNß when discussing new treatment options with MS patients.

Sustained Release Talazoparib Implants for Localized Treatment of BRCA1-deficient Breast Cancer.

Talazoparib, a potent PARP inhibitor, has shown promising clinical and pre-clinical activity by inducing synthetic lethality in cancers with germline Brca1/2 mutations. Conventional oral delivery of Talazoparib is associated with significant off-target effects, therefore we sought to develop new delivery systems in the form of an implant loaded with Talazoparib for localized, slow and sustained release of the drug at the tumor site in Brca1-deficient breast cancer. Poly(lactic-co-glycolic acid) (PLGA) implants (0.8 mm diameter) loaded with subclinical dose (25 or 50 µg) Talazoparib were fabricated and characterized. In vitro studies with Brca1-deficient W780 and W0069 breast cancer cells were conducted to test sensitivity to PARP inhibition. The in vivo therapeutic efficacy of Talazoparib implants was assessed following a one-time intratumoral injection in Brca1Co/Co;MMTV-Cre;p53+/- mice and compared to drug-free implants and oral gavage. Immunohistochemistry studies were performed on tumor sections using PCNA and γ-H2AX staining. Sustained release of Talazoparib was observed over 28 days in vitro. Mice treated with Talazoparib implants showed statistically significant tumor growth inhibition compared to those receiving drug-free implants or free Talazoparib orally. Talazoparib implants were well-tolerated at both drug doses and resulted in less weight loss than oral gavage. PARP inhibition in mice treated with Talazoparib implants significantly increased double-stranded DNA damage and decreased tumor cell proliferation as shown by PCNA and γ-H2AX staining as compared to controls. These results demonstrate that localized and sustained delivery of Talazoparib via implants has potential to provide superior treatment outcomes at sub-clinical doses with minimal toxicity in patients with BRCA1 deficient tumors.

Pegylated interferon beta in the treatment of the Theiler's murine encephalomyelitis virus mouse model of multiple sclerosis.

We evaluated the effects of pegylated-interferonß-1a (pegIFNß) therapy on intrathecal antibody responses, disability progression, and viral load in the CNS in mice infected with the Theiler's virus (TMEV), an animal model of progressive disability in Multiple Sclerosis (MS). The lack of a direct antiviral activity in the CNS, the absence of any effect upon the intrathecal immune response, and the failure to treat disease progression, indicate that the immunomodulatory effects of pegIFNß-1a likely occur in the systemic circulation rather than within the CNS. These results may be relevant to the relative lack of effect of IFNß in progressive MS relative to relapsing MS.

Treatment of Theiler's virus-induced demyelinating disease with teriflunomide.

Teriflunomide is an oral therapy approved for the treatment of relapsing remitting multiple sclerosis (MS), showing both anti-inflammatory and antiviral properties. Currently, it is uncertain whether one or both of these properties may explain teriflunomide's beneficial effect in MS. Thus, to learn more about its mechanisms of action, we evaluated the effect of teriflunomide in the Theiler's encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) model, which is both a viral infection and an excellent model of the progressive disability of MS. We assessed the effects of the treatment on central nervous system (CNS) viral load, intrathecal immune response, and progressive neurological disability in mice intracranially infected with TMEV. In the TMEV-IDD model, we showed that teriflunomide has both anti-inflammatory and antiviral properties, but there seemed to be no impact on disability progression and intrathecal antibody production. Notably, benefits in TMEV-IDD were mostly mediated by effects on various cytokines produced in the CNS. Perhaps the most interesting result of the study has been teriflunomide's antiviral activity in the CNS, indicating it may have a role as an antiviral prophylactic and therapeutic compound for CNS viral infections.

Bromodomain inhibitors, JQ1 and I-BET 762, as potential therapies for pancreatic cancer.

Bromodomain inhibitors (JQ1 and I-BET 762) are a new generation of selective, small molecule inhibitors that target BET (bromodomain and extra terminal) proteins. By impairing their ability to bind to acetylated lysines on histones, bromodomain inhibitors interfere with transcriptional initiation and elongation. BET proteins regulate several genes responsible for cell cycle, apoptosis and inflammation. In this study, JQ1 and I-BET 762 decreased c-Myc and p-Erk 1/2 protein levels and inhibited proliferation in pancreatic cancer cells. The tumor microenvironment is known to play an important role in pancreatic cancer, and these drugs suppressed the production of nitric oxide and a variety of inflammatory cytokines, including IL-6, CCL2, and GM-CSF, in both immune and pancreatic cancer cells in vitro. Notably, the bromodomain inhibitors also reduced protein levels of p-Erk 1/2 and p-STAT3 in mouse models of pancreatic cancer. All of these proteins are essential for tumor promotion, progression and metastasis. In conclusion, the bromodomain inhibitors JQ1 and I-BET 762 targeted and suppressed multiple pathways in pancreatic cancer. I-BET 762 and a number of other bromodomain inhibitors are currently being tested in several clinical trials, making them potentially promising drugs for the treatment of pancreatic cancer, an often-fatal disease.

Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis.

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating disease, with clinical features similar to the progressive forms of multiple sclerosis (MS). The mice show progressive disability with loss of motor and sensory functions, which can be assessed with multiple apparatuses and protocols. Among them, the Rotarod performance test is a very common behavioral test, its advantage being that it provides objective measurements, but it is often used assuming that it is straightforward and simple. In contrast to visual scoring systems used in some models of MS, which are highly subjective, the Rotarod test generates an objective, measurable, continuous variable (i.e., length of time), allowing almost perfect inter-rater concordances. However, inter-laboratory reliability is only achieved if the various testing parameters are replicated. In this manuscript, recommendations of specific testing parameters, such as size, speed, and acceleration of the rod; amount of training given to the animals; and data processing, are presented for the Rotarod test.

The Rexinoids LG100268 and LG101506 Inhibit Inflammation and Suppress Lung Carcinogenesis in A/J Mice.

LG101506 was originally synthesized to overcome some of the undesirable side effects of rexinoids. We compared the anticarcinogenic action of LG101506 and LG100268 and for the first time showed that both drugs are useful for prevention of lung cancer in A/J mice. These molecules markedly reduced tumor number, tumor size, and total tumor burden, when chronically administered to A/J mice that had been initiated with the mutagenic carcinogen, vinyl carbamate. Moreover, LG100268 synergized with the histone deacetylase inhibitor, vorinostat, for prevention of experimental lung cancer and enhanced the effect of carboplatin/paclitaxel for treatment of experimental lung cancer. Both rexinoids diminished the percentage of high-grade, highly malignant adenocarcinomas found at autopsy. In cell culture studies, the rexinoids exhibited potent anti-inflammatory properties at nanoMolar concentrations. These drugs suppressed the ability of lipopolysaccharide to stimulate the synthesis and secretion of nitric oxide and inflammatory cytokines and chemokines, such as IL6, IL1ß, CXCL2, and CSF3, in macrophage-like RAW264.7 cells. The present results suggest that LG100268, LG101506, or a related rexinoid may have useful clinical applications in the field of oncology.

Novel synthetic pyridyl analogues of CDDO-Imidazolide are useful new tools in cancer prevention.

Two new analogues of CDDO-Imidazolide (CDDO-Im), namely 1-[2-Cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]-4(-pyridin-2-yl)-1H-imidazole ("CDDO-2P-Im") and 1-[2-Cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]-4(-pyridin-3-yl)-1H-imidazole ("CDDO-3P-Im") have been synthesized and tested for their potential use as chemopreventive drugs. At nanomolar concentrations, they were equipotent to CDDO-Im for inducing differentiation and apoptosis in U937 leukemia cells. As inflammation and oxidative stress contribute to carcinogenesis, we also assessed their cytoprotective potential. The new compounds suppressed inducible nitric oxide synthase (iNOS) expression in RAW264.7 macrophage-like cells and significantly elevated heme oxygenase-1 (HO-1) and quinone reductase (NQO1) mRNA and protein levels in various mouse tissues in vivo. Most importantly, pharmacokinetic studies performed in vitro in human plasma and in vivo showed that each new analogue was more stable than CDDO-Im. Much higher concentrations of the new derivatives were found in mouse liver, lung, pancreas and kidney after gavage in contrast to CDDO-Im. Because of their better bioavailability and their excellent anti-inflammatory profile in vitro, CDDO-2P-Im and CDDO-3P-Im were tested for prevention in a highly relevant mouse lung cancer model, in which A/J mice develop lung carcinomas after injection of vinyl carbamate, a potent carcinogen. CDDO-2P-Im and CDDO-3P-Im were as effective as CDDO-Im for reducing the size and the severity of the lung tumors.

Dimethyl fumarate and the oleanane triterpenoids, CDDO-imidazolide and CDDO-methyl ester, both activate the Nrf2 pathway but have opposite effects in the A/J model of lung carcinogenesis.

Lung cancer accounts for the highest number of cancer-related deaths in the USA, highlighting the need for better prevention and therapy. Activation of the Nrf2 pathway detoxifies harmful insults and reduces oxidative stress, thus preventing carcinogenesis in various preclinical models. However, constitutive activation of the Nrf2 pathway has been detected in numerous cancers, which confers a survival advantage to tumor cells and a poor prognosis. In our study, we compared the effects of two clinically relevant classes of Nrf2 activators, dimethyl fumarate (DMF) and the synthetic oleanane triterpenoids, CDDO-imidazolide (CDDO-Im) and CDDO-methyl ester (CDDO-Me) in RAW 264.7 mouse macrophage-like cells, in VC1 lung cancer cells and in the A/J model of lung cancer. Although the triterpenoids and DMF both activated the Nrf2 pathway, CDDO-Im and CDDO-Me were markedly more potent than DMF. All of these drugs reduced the production of reactive oxygen species and inhibited nitric oxide production in RAW264.7 cells, but the triterpenoids were 100 times more potent than DMF in these assays. Microarray analysis revealed that only 52 of 99 Nrf2-target genes were induced by all three compounds, and each drug regulated a unique subset of Nrf2 genes. These drugs also altered the expression of other genes important in lung cancer independent of Nrf2. Although all three compounds enhanced the phosphorylation of CREB, only DMF increased the phosphorylation of Akt. CDDO-Me, at either 12.5 or 50mg/kg of diet, was the most effective drug in our lung cancer mouse model. Specifically, CDDO-Me significantly reduced the average tumor number, size and burden compared with the control group (P < 0.05). Additionally, 52% of the tumors in the control group were high-grade tumors compared with only 14% in the CDDO-Me group. Though less potent, CDDO-Im had similar activity as CDDO-Me. In contrast, 61-63% of the tumors in the DMF groups (400-1200mg/kg diet) were high-grade tumors compared with 52% for the controls (P < 0.05). Additionally, DMF significantly increased the average number of tumors compared with the controls (P < 0.05). Thus, in contrast to the triterpenoids, which effectively reduced pathogenesis in A/J mice, DMF enhanced the severity of lung carcinogenesis in these mice. Collectively, these results suggest that although CDDO-Im, CDDO-Me and DMF all activate the Nrf2 pathway, they target distinct genes and signaling pathways, resulting in opposite effects for the prevention of experimental lung cancer.

The PARP inhibitors, veliparib and olaparib, are effective chemopreventive agents for delaying mammary tumor development in BRCA1-deficient mice.

Poly-ADP ribose polymerase (PARP) inhibitors are effective for the treatment of BRCA-deficient tumors. Women with these mutations have an increased risk of developing breast cancer and would benefit from effective chemoprevention. This study examines whether the PARP inhibitors, veliparib and olaparib, delay mammary gland tumor development in a BRCA1-deficient (BRCA1(Co/Co);MMTV-Cre;p53(+/-)) mouse model. In dose de-escalation studies, mice were fed with control, veliparib (100 mg/kg diet), or olaparib (200, 100, 50, or 25 mg/kg diet) continuously for up to 43 weeks. For intermittent dosing studies, mice cycled through olaparib (200 mg/kg diet) for 2 weeks followed by a 4-week rest period on control diet. To examine biomarkers, mice were fed with olaparib using the intermittent dosing regimen and mammary glands were evaluated by immunohistochemistry. In mice treated with veliparib or olaparib (200 mg/kg diet), the average age of the first detectable tumor was delayed by 2.4 and 6.5 weeks, respectively, compared with controls. Olaparib also increased the average lifespan of mice by 7 weeks. In dose de-escalation studies, lower concentrations of olaparib delayed tumor development but were less effective than the highest dose. When fed intermittently, olaparib delayed the onset of the first palpable tumor by 5.7 weeks and significantly reduced proliferation and induced apoptosis in hyperplastic mammary glands. In summary, veliparib and olaparib are effective for delaying tumor development and extending the lifespan of BRCA1-deficient mice, and intermittent dosing with olaparib was as effective as continuous dosing. These results suggest that the use of PARP inhibitors is a promising chemopreventive option.

The combination of the histone deacetylase inhibitor vorinostat and synthetic triterpenoids reduces tumorigenesis in mouse models of cancer.

Novel drugs and drug combinations are needed for the chemoprevention and treatment of cancer. We show that the histone deacetylase inhibitor vorinostat [suberoylanilide hydroxamic acid (SAHA)] and the methyl ester or ethyl amide derivatives of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-Me and CDDO-Ea, respectively) cooperated to inhibit the de novo synthesis of nitric oxide in RAW 264.7 macrophage-like cells and in primary mouse peritoneal macrophages. Additionally, SAHA enhanced the ability of synthetic triterpenoids to delay formation of estrogen receptor-negative mammary tumors in MMTV-polyoma middle T (PyMT) mice. CDDO-Me (50 mg/kg diet) and SAHA (250 mg/kg diet) each significantly delayed the initial development of tumors by 4 (P < 0.001) and 2 (P < 0.05) weeks, respectively, compared with the control group in the time required to reach 50% tumor incidence. CDDO-Ea (400 mg/kg diet), as a single agent, did not delay tumor development. The combination of either triterpenoid with SAHA was significantly more potent than the individual drugs for delaying tumor development, with a 7 week (P < 0.001) delay before 50% tumor incidence was reached. SAHA, alone and in combination with CDDO-Me, also significantly (P < 0.05) inhibited the infiltration of tumor-associated macrophages into the mammary glands of PyMT mice and levels of the chemokine macrophage colony-stimulating factor in primary PyMT tumor cells. In addition, SAHA and the synthetic triterpenoids cooperated to suppress secreted levels of the pro-angiogenic factor matrix metalloproteinase-9. Similar results were observed in mouse models of pancreatic and lung cancer. At concentrations that were anti-inflammatory, SAHA had no effect on histone acetylation. These studies suggest that both SAHA and triterpenoids effectively delay tumorigenesis, thereby demonstrating a promising, novel drug combination for chemoprevention.

CDDO-methyl ester delays breast cancer development in BRCA1-mutated mice.

The breast cancer-associated gene 1 (BRCA1) is the most frequently mutated tumor suppressor gene in familial breast cancers. Mutations in BRCA1 also predispose to other types of cancers, pointing to a fundamental role of this pathway in tumor suppression and emphasizing the need for effective chemoprevention in these high-risk patients. Because the methyl ester of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-Me) is a potent chemopreventive agent, we tested its efficacy in a highly relevant mouse model of BRCA1-mutated breast cancer. Beginning at 12 weeks of age, Brca1(Co/Co); MMTV-Cre;p53(+/-) mice were fed powdered control diet or diet containing CDDO-Me (50 mg/kg diet). CDDO-Me significantly (P < 0.05) delayed tumor development in the Brca1-mutated mice by an average of 5.2 weeks. We also observed that levels of ErbB2, p-ErbB2, and cyclin D1 increased in a time-dependent manner in the mammary glands in Brca1-deficient mice, and CDDO-Me inhibited the constitutive phosphorylation of ErbB2 in tumor tissues from these mice. In BRCA1-deficient cell lines, the triterpenoids directly interacted with ErbB2, decreased constitutive phosphorylation of ErbB2, inhibited proliferation, and induced G(0)-G(1) arrest. These results suggest that CDDO-Me has the potential to prevent BRCA1-mutated breast cancer.

Synthetic triterpenoids prolong survival in a transgenic mouse model of pancreatic cancer.

Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States and is nearly always fatal. Whereas early detection offers the most promising approach for reducing the mortality of this disease, there is still a need to develop effective drugs for the prevention and treatment of pancreatic cancer. We tested two promising classes of noncytotoxic drugs, synthetic oleanane triterpenoids and rexinoids, for the prevention of carcinogenesis in the highly relevant LSL-Kras(G12D/+);LSL-Trp53(R127H/+);Pdx-1-Cre (KPC) mouse model of pancreatic cancer. KPC transgenic mice closely recapitulate the genetic mutations, clinical symptoms, and histopathology found in human pancreatic cancer. Beginning at 4 weeks of age, mice were fed powdered control diet or a diet containing the triterpenoids CDDO-methyl ester (CDDO-Me) or CDDO-ethyl amide, the rexinoid LG100268 (LG268), or the combination, until the mice displayed overt symptoms of pancreatic cancer. CDDO-Me, LG268, the combination of CDDO-Me and LG268, and the combination of CDDO-ethyl amide and LG268, all significantly (P < 0.05) increased survival in the KPC mice by 3 to 4 weeks. Recent studies have shown that gemcitabine, the current standard of care for human pancreatic cancer, does not extend survival in KPC mice. In cell lines developed from the KPC mice, the triterpenoids directly interact with both signal transducer and activator of transcription 3 and IκB kinase (IKK) to decrease constitutive interleukin-6 secretion, inhibit constitutive signal transducer and activator of transcription 3 phosphorylation, and block the degradation of IκBα when challenged with tumor necrosis factor α. These results suggest that oleanane triterpenoids and rexinoids have the potential to prevent pancreatic cancer.