Rutin restrains the growth and metastasis of mouse breast cancer cells by regulating the microRNA-129-1-3p-mediated calcium signaling pathway.

Breast cancer is a common malignancy that is highly lethal. Due to the poor prognosis, more effective and efficient treatment methods are urgently needed. Rutin (RUT) is a traditional Chinese medicine reported to have a variety of pharmacological properties, including anticancer properties. However, the effects of RUT on breast cancer and its underlying molecular mechanism of action remain unclear. In the present study, we observed a significant downregulation of microRNA (miR)-129-1-3p in mouse breast cancer cells (4T1) compared with the expression in mouse normal breast epithelial cells (HC11). We also found that RUT could increase the expression of miR-129-1-3p in 4T1 cells and suppress cell proliferation. To elucidate the molecular mechanism of action of RUT, miR-129-1-3p mimics and its inhibitor were transfected into 4T1 cells. miR-129-1-3p overexpression could inhibit the proliferation, invasion, migration, and calcium overload of mouse breast cancer cells and also enhance apoptosis, whereas miR-129-1-3p knockdown had the opposite effects. Taken together, cell-based experiments indicated that RUT restrains the growth of mouse breast cancer cells by regulating the miR-129-1-3p/Ca2+ signaling pathway. This study also revealed the inhibitory effect of RUT on breast cancer cells at the noncoding RNA level and provided a theoretical foundation for the application of RUT as a drug to inhibit tumor growth.

Mammary tumors compromise time-of-day differences in hypothalamic gene expression and circadian behavior and physiology in mice.

Circadian rhythms influence various aspects of biology, including hormonal, immunological, and behavioral processes. These 24-hour oscillations are necessary to optimize cellular functions and to synchronize these processes with the environment. Breast cancer patients and survivors frequently report disruptions in circadian oscillations that adversely affect quality-of-life, including fragmented sleep-wake cycles and flattened cortisol rhythms, which are associated with negative behavioral comorbidities (e.g., fatigue). However, the potential causal role of tumor biology in circadian dysregulation has not been investigated. Here, we examined the extent to which sham surgery, non-metastatic mammary tumors, or mammary tumor removal in mice disrupts circadian rhythms in brain clock gene expression, locomotor behavior (free-running and entrained), and physiological rhythms that have been associated with cancer behavioral comorbidities. Tumors and tumor resection altered time-of-day differences in hypothalamic expression of eight circadian-regulated genes. The onset of activity in entrained running behavior was advanced in tumor-bearing mice, and the amplitude of free-running rhythms was increased in tumor-resected mice. Tumors flattened rhythms in circulating corticosterone and Ly6cHi monocytes which were largely restored by surgical tumor resection. This work implies that tumors alone may directly impact central and/or peripheral circadian rhythmicity in breast cancer patients, and that these effects may persist in cancer survivors, potentially contributing to behavioral comorbidities.

Anticancer Effects of Gleditsia sinensis Extract in Rats Transplanted With Hepatocellular Carcinoma Cells.

The thorns of Gleditsia sinensis have been historically used in Chinese medicine and are considered one of the fundamental therapeutic herbs. Its anticancer effects are currently being explored. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and still requires the development of new drugs with higher efficiency. By using a rat HCC model implanted with cancerous Walker-256 cells, the therapeutic effects of G. sinensis extract (GSE) were assessed, as well as its regulatory effects on miRNAs. GSE significantly restored liver morphology and dramatically induced cell apoptosis in HCC rats. In addition, miR-21/181b/183 was upregulated in the HCC liver, and the elevation of these miRNAs could be alleviated by both GSE and sorafenib. PTEN/TIMP3/PDCD4 downregulation was consistent with the targets of miR-21/181b/183 in the HCC liver, and the alteration of these target genes was restored by both GSE and sorafenib. TIMP3 effects on MMP-2/9 expression were also determined. Our present findings indicate the potential of GSE in HCC treatment, and expand the understanding of miRNA-related mechanisms in the anticancer effects of GSE.

Endogenously Synthesized n-3 Polyunsaturated Fatty Acids in Pregnant fat-1 Mice Decreases Mammary Cancer Risk of Female Offspring by Regulating Expression of Long Noncoding RNAs.

SCOPE: The present study investigates the precise mechanism by which maternal n-3 PUFAs decrease mammary cancer risk of offspring in terms of epigenetics. METHODS AND RESULTS: Transgenic fat-1 and wild-type C57BL/6J littermates are fed an n-6 PUFAs diet during pregnancy. Wild-genotype offspring of fat-1 mothers (fat-1 group) are compared with wild-genotype offspring of C57BL/6J mothers (control group) in breast cancer risk. Fat-1 group shows a significantly lower tumor incidence and smaller tumor volume compared with control group. n-3 PUFAs in fat-1 mothers change the expression of long noncoding RNA (lncRNA, 53 upregulated and 45 downregulated) in mammary glands of offspring. The lncRNA changes are associated with the changes of mRNA in multiple oncogenic signaling pathways, especially NF-κB, Jak-STAT, and MAPK pathways. Expression of key protein in these pathways, namely p65, p60, STAT3, Jak1, and p38, are significantly inhibited in fat-1 group. In line with these results, reduced proliferation and increased apoptosis are also observed in mammary epithelial of fat-1 group than control group. CONCLUSION: The anticancer effect of maternal n-3 PUFAs is related to the regulation of lncRNA expression, which can further regulate the susceptibility of offspring to breast cancer.

Dietary intake of iodine-enriched eggs decreases the incidence of mouse mammary tumors caused by the activated ErbB2 oncogene.

Human epigenetic studies suggest that consumption of seaweed prevents mammary cancer, which possibly is explained by iodine daily intake. In this study, we evaluated the efficacy of dietary intake of iodine-enriched eggs on mammary tumor incidence caused by the expression of activated type ErbB2. Female transgenic mice were divided into three groups, and fed a basic diet, a diet supplemented with ordinary eggs, or with iodine-enriched eggs. The number of mammary tumors greater than 5 mm in diameter was recorded in mice at 6 months of age. We report that the average number of mammary tumors per mouse was significantly lower in the iodine-enriched egg-added diet group than in either the basic diet or ordinary egg diet groups. These results indicate that iodine intake through livestock-derived products can reduce the incidence of mammary cancers caused by the expression of activated type ErbB2.

Involvement of Prokineticin 2-expressing Neutrophil Infiltration in 5-Fluorouracil-induced Aggravation of Breast Cancer Metastasis to Lung.

Adjuvant chemotherapy is used for human breast cancer patients, even after curative surgery of primary tumor, to prevent tumor recurrence primarily as a form of metastasis. However, anticancer drugs can accelerate metastasis in several mouse metastasis models. Hence, we examined the effects of postsurgical administration with 5-fluorouracil (5-FU), doxorubicin, and cyclophosphamide, on lung metastasis process, which developed after the resection of the primary tumor arising from the orthotopic injection of a mouse triple-negative breast cancer cell line, 4T1. Only 5-FU markedly increased the numbers and sizes of lung metastasis foci, with enhanced tumor cell proliferation and angiogenesis as evidenced by increases in Ki67-positive cell numbers and CD31-positive areas, respectively. 5-FU-mediated augmented lung metastasis was associated with increases in intrapulmonary neutrophil numbers and expression of neutrophilic chemokines, Cxcl1 and Cxcl2 in tumor cells, with few effects on intrapulmonary T-cell or macrophage numbers. 5-FU enhanced Cxcl1 and Cxcl2 expression in 4T1 cells in a NFκB-dependent manner. Moreover, the administration of a neutrophil-depleting antibody or a Cxcr2 antagonist, SB225002, significantly attenuated 5-FU-mediated enhanced lung metastasis with depressed neutrophil infiltration. Furthermore, infiltrating neutrophils and 4T1 cells abundantly expressed prokineticin-2 (Prok2) and its receptor, Prokr1, respectively. Finally, the administration of 5-FU after the resection of the primary tumor failed to augment lung metastasis in the mice receiving Prokr1-deleted 4T1 cells. Collectively, 5-FU can enhance lung metastasis by inducing tumor cells to produce Cxcl1 and Cxcl2, which induced the migration of neutrophils expressing Prok2 with a capacity to enhance 4T1 cell proliferation. Mol Cancer Ther; 17(7); 1515-25. ©2018 AACR.

1,25-Dihydroxy vitamin D prevents tumorigenesis by inhibiting oxidative stress and inducing tumor cellular senescence in mice.

Human epidemiological studies suggest that 1,25(OH)2 D3 deficiency might increase cancer incidence, but no spontaneous tumors have been reported in mice lacking 1,25(OH)2 D3 or deficient in its receptor. In our study, we detected, for the first time, diverse types of spontaneous tumors in l,25(OH)2 D3 deficient mice more than 1 year of age. This was associated with increased oxidative stress, cellular senescence and senescence-associated secretory phenotype molecules, such as hepatocyte growth factor, mediated via its receptor c-Met. Furthermore, 1,25(OH)2 D3 prevented spontaneous tumor development. We also demonstrated that l,25(OH)2 D3 deficiency accelerates allograft tumor initiation and growth by increasing oxidative stress and DNA damage, activating oncogenes, inactivating tumor suppressor genes, stimulating malignant cell proliferation and inhibiting their senescence; in contrast, supplementation with exogenous l,25(OH)2 D3 or antioxidant, or knock-down of the Bmi1 or c-Met oncogene, largely rescued the phenotypes of allograft tumors. Results from our study suggest that 1,25(OH)2 D3 deficiency enhances tumorigenesis by increasing malignant cell oxidative stress and DNA damage, stimulating microenvironmental cell senescence and a senescence-associated secretory phenotype, and activating oncogenes and inactivating tumor suppressor genes, thus increasing malignant cell proliferation. Our study provides direct evidence supporting the role of vitamin D deficiency in increasing cancer incidence. Conversely, 1,25(OH)2 D3 prevented spontaneous tumor development, suggesting that this inhibitory effect prevents the initiation and progression of tumorigenesis, thus provides a mechanistic basis for 1,25(OH)2 D3 to prevent tumorigenesis in an aging organism.

Fucose-containing fraction of Ling-Zhi enhances lipid rafts-dependent ubiquitination of TGFß receptor degradation and attenuates breast cancer tumorigenesis.

Ganoderma lucidum exerts antitumor activity, but the mechanism of G. lucidum polysaccharides on cancer is unclear. Here, we demonstrated that a fucose-containing fraction of Ling-Zhi (FFLZ) reduced tumor size and suppressed metastasis in vivo. Furthermore, FFLZ inhibited breast cancer cell migration and altered the epithelial-to-mesenchymal transition (EMT) phenotype. Transforming growth factor-ß receptor (TGFR) pathways act as key mediators to promote tumor progression and metastasis. We found that FFLZ down-regulated TGFR and downstream signaling pathways, including the phosphorylation of Smad2/3 and the expression of Smad4. In an investigation of the underlying mechanisms, we found that FFLZ enhanced the Smurf2-dependent ubiquitination of TGFR by disrupting the balance of the lipid rafts, promoted the "re-localization" of the TGFR to the caveolae, and facilitated the degradation of TGFR. Together, our data indicated that FFLZ is associated with the inhibition of EMT and the prevention of metastasis by promoting ubiquitination-dependent TGFR degradation and abolishing TGFR signaling pathways. Moreover, the combination of FFLZ and trastuzumab synergistically inhibited the viability of certain trastuzumab-resistant human breast cancer cells. In summary, our current findings indicate that FFLZ is a potential therapeutic or dietary supplemental agent for cancer patients and that it functions via the caveolin-1/Smad7/Smurf2-dependent ubiquitin-mediated degradation of TGFR.

The Role of Dietary Extra Virgin Olive Oil and Corn Oil on the Alteration of Epigenetic Patterns in the Rat DMBA-Induced Breast Cancer Model.

Disruption of epigenetic patterns is a major change occurring in all types of cancers. Such alterations are characterized by global DNA hypomethylation, gene-promoter hypermethylation and aberrant histone modifications, and may be modified by environment. Nutritional factors, and especially dietary lipids, have a role in the etiology of breast cancer. Thus, we aimed to analyze the influence of different high fat diets on DNA methylation and histone modifications in the rat dimethylbenz(a)anthracene (DMBA)-induced breast cancer model. Female Sprague-Dawley rats were fed a low-fat, a high corn-oil or a high extra-virgin olive oil (EVOO) diet from weaning or from induction with DMBA. In mammary glands and tumors we analyzed global and gene specific (RASSF1A, TIMP3) DNA methylation by LUMA and bisulfite pyrosequencing assays, respectively. We also determined gene expression and enzymatic activity of DNA methyltransferases (DNMT1, DNMT3a and DNMT3b) and evaluated changes in histone modifications (H3K4me2, H3K27me3, H4K20me3 and H4K16ac) by western-blot. Our results showed variations along time in the global DNA methylation of the mammary gland displaying decreases at puberty and with aging. The olive oil-enriched diet, on the one hand, increased the levels of global DNA methylation in mammary gland and tumor, and on the other, changed histone modifications patterns. The corn oil-enriched diet increased DNA methyltransferase activity in both tissues, resulting in an increase in the promoter methylation of the tumor suppressor genes RASSF1A and TIMP3. These results suggest a differential effect of the high fat diets on epigenetic patterns with a relevant role in the neoplastic transformation, which could be one of the mechanisms of their differential promoter effect, clearly stimulating for the high corn-oil diet and with a weaker influence for the high EVOO diet, on breast cancer progression.

Effects of Ginkgo biloba on chemically-induced mammary tumors in rats receiving tamoxifen.

BACKGROUND: Ginkgo biloba extract (GbE) is used extensively by breast cancer patients undergoing treatment with Tamoxifen (TAM). Thus, the present study investigated the effects of GbE in female Sprague-Dawley (SD) rats bearing chemically-induced mammary tumors and receiving TAM. METHODS: Animals bearing mammary tumors (≥1 cm in diameter) were divided into four groups: TAM [10 mg/kg, intragastrically (i.g.)], TAM plus GbE [50 and 100 mg/kg, intraperitoneally (i.p.)] or an untreated control group. After 4 weeks, the therapeutic efficacy of the different treatments was evaluated by measuring the tumor volume (cm(3)) and the proportions of each tumor that were alive, necrotic or degenerative (mm(2)). In addition, labeling indexes (LI%) were calculated for cell proliferation (PCNA LI%) and apoptosis (cleaved caspase-3 LI%), expression of estrogen receptor-alpha (ER-α) and p63 biomarkers. RESULTS: Overall, the tumor volume and the PCNA LI% within live tumor areas were reduced by 83% and 99%, respectively, in all TAM-treated groups when compared to the untreated control group. GbE treatment (100 mg/kg) reduced the proportions of live (24.8%) and necrotic areas (2.9%) (p = 0.046 and p = 0.038, respectively) and significantly increased the proportion of degenerative areas (72.9%) (p = 0.004) in mammary tumors when compared to the group treated only with TAM. The expression of ER-α, p63 and cleaved caspase-3 in live tumor tissues was not modified by GbE treatment. CONCLUSIONS: Co-treatment with 100 mg/kg GbE presented a slightly beneficial effect on the therapeutic efficacy of TAM in female SD rats bearing mammary tumors.

Effect of annatto-tocotrienols supplementation on the development of mammary tumors in HER-2/neu transgenic mice.

Tocotrienols (T3), the lesser known isomers of vitamin E, have been reported to possess anticancer activity both in in vitro and in vivo experimental models of rodents transplanted with parental tumors or treated with carcinogens. We investigated the effects of dietary supplementation with annatto-T3 (90% δ-T3 and 10% γ-T3) on the spontaneous development of mammary tumors in HER-2/neu transgenic mice. Underlying mechanisms of the antitumor effect were evaluated by studying apoptosis, senescent-like growth arrest, immune modulation, oxidative effect and the expression of HER-2/neu in tumoral mammary glands of transgenic mice and in vitro in human and mice tumor cell lines. Annatto-T3 supplementation delayed the development of mammary tumors, reducing the number and size of mammary tumor masses and those of lung metastases. In annatto-T3-supplemented mice, both apoptosis and senescent-like growth arrest of tumor cells were increased in mammary glands while no immune modulation was observed. In vitro, a dose-dependent inhibition of cell growth, increased apoptosis and senescent-like growth arrest and a time-dependent accumulation of reactive oxygen species were observed in tumor cells treated with annatto-T3 or purified δ-T3. Annatto-T3 reduced both HER-2/neu mRNA and p185(HER-2/neu) protein in tumors and in tumor cell lines. The results show that the antitumor effect of annatto-T3 supplementation in HER-2/neu transgenic mice is mainly related to the direct induction of oxidative stress, senescent-like growth arrest and apoptosis of tumor cells rather than to an immune modulation.

Heterologous expression and functional characterization of matrix metalloproteinase-11 from canine mammary tumor.

Matrix metalloproteinases (MMPs) are reported to be involved in tumor growth, apoptosis, angiogenesis, invasion, and development of metastases. These are zinc containing metalloproteases, known for their role in extracellular matrix degradation. MMP-11 (stromelysin3) is reported to be highly expressed in breast cancer, therefore it may act as marker enzyme for breast cancer progression. The present work was carried out to produce recombinant canine (Canis lupus familiaris) MMP-11 lacking the signal and propeptide in E. coli by optimizing its expression and purification in biologically active form and to functionally characterize it. A bacterial protein expression vector pPROEX HTc was used. The MMP-11 mature peptide encoding gene was successfully cloned and expressed in E. coli and the purified recombinant enzyme was found to be functionally active. The recombinant enzyme exhibited caseinolytic activity and could be activated by Trypsin and 4-Amino phenyl mercuric acetate (APMA). However Ethylene diamine tertra acetate (EDTA) inhibited the enzyme's caseinolytic activity. The recombinant enzyme degraded extracellular matrix constituents and facilitated migration of MDCK (Madin-Darby canine kidney) cells through BD Biocoat Matrigel invasion chambers. These results suggest that in vivo MMP-11 could play a significant role in the turnover of extracellular matrix constituents.

Lack of ABCG2 shortens latency of BRCA1-deficient mammary tumors and this is not affected by genistein or resveratrol.

In addition to their role in drug resistance, the ATP-binding cassette (ABC) transporters ABCG2 and ABCB1 have been suggested to protect cells from a broad range of substances that may foster tumorigenesis. Phytoestrogens or their metabolites are substrates of these transporters and the influence of these compounds on breast cancer development is controversial. Estrogen-like properties might accelerate tumorigenesis on the one hand, whereas their proposed health-protective properties might antagonize tumorigenesis on the other. To address this issue, we used a newer generation mouse model of BRCA1-mutated breast cancer and examined tumor latency in K14cre;Brca1(F/F); p53(F/F), Abcb1a/b(-/-);K14cre;Brca1(F/F); p53(F/F), or Abcg2(-/-);K14cre;Brca1(F/F); p53(F/F) animals, fed with genistein- or resveratrol-supplemented diets. Ovariectomized K14cre;Brca1(F/F); p53(F/F) animals were included to evaluate whether any estrogen-mimicking effects can restore mammary tumor development in the absence of endogenous estrogens. Compared with the ABC transporter proficient model, ABCG2-deficient animals showed a reduced median tumor latency of 17.5 days (P < 0.001), whereas no significant difference was observed for ABCB1-deficient animals. Neither genistein nor resveratrol altered this latency reduction in Abcg2(-/-);K14cre;Brca1(F/F); p53(F/F) animals. Ovariectomy resulted in nearly complete loss of mammary tumor development, which was not restored by genistein or resveratrol. Our results show that ABCG2 contributes to the protection of genetically instable epithelial cells against carcinogenesis. Diets containing high levels of genistein or resveratrol had no effect on mammary tumorigenesis, whether mice were lacking ABCG2 or not. Because genistein and resveratrol only delayed skin tumor development of ovariectomized animals, we conclude that these phytoestrogens are no effective modulators of mammary tumor development in our mouse model.

Atorvastatin modulates anti-proliferative and pro-proliferative signals in Her2/neu-positive mammary cancer.

The widely used anticholesterolemic drugs statins decrease the synthesis of cholesterol and the isoprenylation and activity of small G-proteins such as Ras and Rho, the effectors of which are often critical in cell proliferation. Thanks to this property, it has been hypothesized that statins may have anti-tumor activities. We investigated this issue in BALB-neuT mice, which developed Her2/neu-positive mammary cancers with 100% penetrance, and in TUBO cells, a cell line established from these tumors. Contrary to the mammary glands of BALB/c mice, the tumor tissue from BALB-neuT animals had constitutively activated Ras and ERK1/2. These were reduced by the oral administration of atorvastatin, but the statin did not prevent tumor growth in mice nor reduce the proliferation of TUBO cells, although it lowered the activity of mevalonate pathway and Ras/ERK1/2 signaling. By decreasing the mevalonate pathway-derived metabolite geranylgeranyl pyrophosphate and the RhoA/RhoA kinase signaling, atorvastatin activated NF-κB, that sustained cell proliferation. Unexpectedly Her2-positive cells were much more sensitive to the inhibition of RhoA-dependent pathways than to the suppression of Ras-dependent pathways elicited by atorvastatin. Only the simultaneous inhibition of RhoA/RhoA-kinase/NF-κB and Ras/ERK1/2 signaling allowed the statin to decrease tumor cell proliferation. Our study demonstrates that Her2-positive mammary cancers have redundant signals to sustain their proliferation and shows that statins simultaneously reduce the pro-proliferative Ras/ERK1/2 axis and activate the pro-proliferative RhoA/RhoA-kinase/NF-κB axis. The latter event dissipates the antitumor efficacy that may arise from the former one. Only the association of statins and NF-κB-targeted therapies efficiently decreased proliferation of tumor cells.

Anti-/pro-apoptotic effects of hesperetin against 7,12-dimetylbenz(a)anthracene-induced alteration in animals.

This study investigated the anticancer effects of hesperetin on 7,12-dimethylbenz(a)anthracene (DMBA)-treated animals and explored its anticancer mechanism. The experiment consisted of two parts. First, Sprague-Dawley rats were given hesperetin daily at a dose of 50 mg/kg for 8 weeks after a single dose of DMBA (100 mg/kg). As controls, rats were divided into vehicle alone and DMBA alone groups. Secondly, ICR mice were given hesperetin daily at a dose of 10 and 50 mg/kg BW/day for 7 weeks before a single dose of DMBA (34 mg/kg/week). In rats with DMBA-induced mammary gland tumors, hesperetin pretreatment significantly reduced the tumor burden and PCNA overexpression. The administration of hesperetin significantly inhibited mammary gland carcinoma from developing by restoring the decreased Bcl-2 and increased Bax expression. By contrast, in the livers of mice treated with DMBA, obvious DNA fragmentation was observed. Moreover, apoptosis-related gene expression in the livers of the mice differed from that in mammary gland carcinomas in rats. These changes were restored in mice treated with hesperetin, indicating the inhibition of apoptosis. Based on these results, hesperetin may act not only as a proapoptotic agent, but also as an antiapoptotic agent, depending on the circumstance.

In vivo effect of alpha-bisabolol, a nontoxic sesquiterpene alcohol, on the induction of spontaneous mammary tumors in HER-2/neu transgenic mice.

Breast cancer represents the most commonly diagnosed invasive malignancy in pre- and postmenopausal women in both developed and underdeveloped countries. Taking into account that treatment options, including surgery, have not been able to deal with the growing incidence of breast malignancy, it is required to develop mechanism-based novel agents for its prevention. Wide interest in some natural compounds as antiinflammatory agents and as alternative to the traditional medicines is increasing because they do not provoke any adverse effects and are effective in multiple organs, alpha-Bisabolol (BISA), a small oily sesquiterpene alcohol, was reported as chemopreventive agent in induced rat mammary carcinogenesis. The aim of the present study is to investigate the role played by two doses of BISA (via intramammary infusion) on the induction and development of mammary tumor in HER-2/neu transgenic mice as well as the BISA effect after tumor surgical resection. The main data show that (a) optimal dosage of BISA is 10 mg/mouse rather than 3.6 mg/mouse with no adverse effects (e.g., alopecia); (b) the number of the palpable tumor masses decreases in mice treated with 10 mg/mouse of BISA; (c) mice after surgical resection of the primary tumor and treatment with BISA (10 mg) are free from tumor for more weeks, after the surgical treatment; (d) using array analysis, some genes implicated in carcinogenesis mechanisms (NF-kappaBia, Map2k, Mapkl4, and HER2/ neu), angiogenesis process (Fgf), and inhibition of apoptosis (Birc5) are differently regulated after BISA treatment, with a downregulation of the HER2/neu as well as of Fgf and Birc5 genes; (e) the NK cell cytotoxicity increases in tumor-treated mice, especially after the removal of the first tumor mass. Such effectiveness could be important to achieve goals for a possible combination of BISA to conventional therapies in breast cancer and to tumor surgical removal (adjuvant therapy), as suggested for other sesquiterpene analogs.

Sensitivity and acquired resistance of BRCA1;p53-deficient mouse mammary tumors to the topoisomerase I inhibitor topotecan.

There is no tailored therapy yet for human basal-like mammary carcinomas. However, BRCA1 dysfunction is frequently present in these malignancies, compromising homology-directed DNA repair. This defect may serve as the tumor's Achilles heel and make the tumor hypersensitive to DNA breaks. We have evaluated this putative synthetic lethality in a genetically engineered mouse model for BRCA1-associated breast cancer, using the topoisomerase I (Top1) poison topotecan as monotherapy and in combination with poly(ADP-ribose) polymerase inhibition by olaparib. All 20 tumors tested were topotecan sensitive, but response heterogeneity was substantial. Although topotecan increased mouse survival, all tumors eventually acquired resistance. As mechanisms of in vivo resistance, we identified overexpression of Abcg2/Bcrp and markedly reduced protein levels of the drug target Top1 (without altered mRNA levels). Tumor-specific genetic ablation of Abcg2 significantly increased overall survival of topotecan-treated animals (P < 0.001), confirming the in vivo relevance of ABCG2 for topotecan resistance in a novel approach. Despite the lack of ABCG2, a putative tumor-initiating cell marker, none of the 11 Abcg2(-/-);Brca1(-/-);p53(-/-) tumors were eradicated, not even by the combination topotecan-olaparib. We find that olaparib substantially increases topotecan toxicity in this model, and we suggest that this might also happen in humans.

Immunological effects of yeast- and mushroom-derived beta-glucans.

Glucans have a long history as nonspecific biological modulators. We compared the effects of three different glucans on immune reactions. Using two different administrations (intraperitoneal and oral) and two different animal models, we showed that yeast-derived Betamune (Biorigin, São Paulo, Brazil) caused significant stimulation of phagocytic activity as well as potentiation of synthesis and release of interleukin (IL)-1, IL-2, IL-4, IL-6, IL-8, IL-13, and tumor necrosis factor-alpha. In addition, Betamune inhibited growth of tumor cells in vivo and affected expression of several important genes in breast cancer cells. Compared to adult mice, young animals showed different sensitivity to glucan action.

OX40 agonist therapy enhances CD8 infiltration and decreases immune suppression in the tumor.

Acquisition of full T-cell effector function and memory differentiation requires appropriate costimulatory signals, including ligation of the costimulatory molecule OX40 (TNFRSF4, CD134). Tumors often grow despite the presence of tumor-specific T cells and establish an environment with weak costimulation and immune suppression. Administration of OX40 agonists has been shown to significantly increase the survival of tumor-bearing mice and was dependent on the presence of both CD4 and CD8 T cells during tumor-specific priming. To understand how OX40 agonists work in mice with established tumors, we developed a model to study changes in immune cell populations within the tumor environment. We show here that systemic administration of OX40 agonist antibodies increased the proportion of CD8 T cells at the tumor site in three different tumor models. The function of the CD8 T cells at the tumor site was also increased by administration of OX40 agonist antibody, and we observed an increase in the proportion of antigen-specific CD8 T cells within the tumor. Despite decreases in the proportion of T regulatory cells at the tumor site, T regulatory cell function in the spleen was unaffected by OX40 agonist antibody therapy. Interestingly, administration of OX40 agonist antibody caused significant changes in the tumor stroma, including decreased macrophages, myeloid-derived suppressor cells, and decreased expression of transforming growth factor-beta. Thus, therapies targeting OX40 dramatically changed the tumor environment by enhancing the infiltration and function of CD8 T cells combined with diminished suppressive influences within the tumor.

High corn oil and high extra virgin olive oil diets have different effects on the expression of differentiation-related genes in experimental mammary tumors.

Dietary lipids can modify the clinical behavior and morphological features of experimental breast tumors. We previously demonstrated that a high corn oil diet has a tumor-enhancing effect in 7,12-dimethylbenz(alpha)anthracene (DMBA)-induced rat mammary adenocarcinomas, whereas a high olive oil diet acts as a negative modulator of carcinogenesis. In this study, we investigated whether these high fat diets modulate the expression of genes related to differentiation. Rats were induced with DMBA and fed a low fat diet, a high corn oil diet, a high olive oil diet, or both high fat diets. The expression levels of the mammary differentiation biomarkers alpha-casein, beta-casein and transferrin and of beta-actin and its transporter zipcode binding protein 1 (ZBP1) were analyzed by Northern and/or Western blot in the mammary adenocarcinomas. The high fat diets did not induce changes in the expression of caseins, while transferrin expression was increased as a result of the high olive oil diet. beta-actin mRNA levels were higher in the high fat diet groups, though no changes in the protein levels were observed. The expression of ZBP1, a protein reported as having a role in carcinogenesis, was significantly increased by the high corn oil diet. These results suggest that in this model caseins are not good biomarkers of the changes in tumor morphological differentiation conferred by the high fat diets. The modulation of transferrin and ZBP1 expression by the high olive oil and the high corn oil diets could be one of the mechanisms by which such diets have a different influence on mammary carcinogenesis.