Rat Mammary Tumors Research Articles

Docosahexaenoic acid reduces sterol regulatory element binding protein-1 and fatty acid synthase expression and inhibits cell proliferation by inhibiting pAkt signaling in a human breast cancer MCF-7 cell line

BackgroundFatty acid synthase (FASN), the major enzyme in de novo fatty acid synthesis, is highly expressed in breast cancer and its expression is reduced by polyunsaturated fatty acids (PUFAs) in liver. We previously found a positive association between rat mammary tumor levels of the n-6 PUFA arachidonic acid (AA) and tumor weight. We examined the roles of the major n-3 PUFA, docosahexaenoic acid (DHA, 22:6n-3), and the major n-6 PUFA, AA, in FASN expression in, and proliferation of, human breast cancer MCF-7 cells.MethodsThe cells were treated for 48 h with BSA or 60 μM BSA-bound DHA, AA, or oleic acid (OA, 18:1n-9), then were incubated with or without estradiol or insulin. Western blot and 3H–thymidine incorporation assay were used to determine the role of DHA on FASN regulation and MCF-7 cell proliferation.ResultsDHA, but neither AA nor OA, inhibits estradiol-induced and insulin-induced expression of the precursor of sterol regulatory element binding protein-1 (p-SREBP-1), its mature form (m-SREBP-1), and FASN. Estradiol or insulin stimulation increased the pAkt/Akt and pS6/S6 ratios, expression of p-SREBP-1, m-SREBP-1, and FASN, and cell proliferation, and these effects were decreased by DHA. The DHA-induced decrease in FASN expression resulted from reduced pAkt/Akt signaling and not pERK1/2/ERK1/2 signaling. In addition, DHA enhanced the inhibitory effect of LY294002 on pAkt signaling and expression of p-SREBP-1, m-SREBP-1, and FASN. However, addition of rapamycin, an inhibitor of the mTOR signaling pathways, 1 h before addition of estradiol or insulin increased the pAkt/Akt ratio and FASN expression, and this effect was inhibited by addition of DHA 48 h before rapamycin.ConclusionWe conclude that, in MCF-7 cells, DHA inhibits pAKT signaling and thus expression of p-SREBP-1, m-SREBP-1, and FASN and cell proliferation.

In vivo anti-tumor efficacy of Croton oblongifolius in DMBA induced mammary tumor in rats

The present study was designed to evaluate anti-tumor activity of the methanolic extract of stem bark of Croton oblongifolius in Sprague Dawley rats. The tumor was induced in rats by DMBA given orally and intramammarily and challenging with plant extract. After obtaining suitable mass of tumor, the extract was gavaged to rats @ 200, 500 and 800 mg/kg which showed reduction in mammary tumor volume in dose-dependent manner, which was supported by histopathological observations of the treatment groups.

Immunomodulatory effects of a bioactive fraction of Strobilanthes crispus in NMU-induced rat mammary tumor model

Ethnopharmacological relevanceStrobilanthes crispus Blume is traditionally consumed among local Malay and indigenous communities for the treatment of cancer and other ailments such as gastrointestinal disorders, inflammatory wounds of snake bite and immune system activation amongst others. We previously demonstrated that a bioactive fraction of S. crispus leaves (F3) was cytotoxic to breast cancer cells in vitro and inhibited tumor growth in N-methyl-N-nitrosourea (NMU)-induced breast cancer rat model. F3 also normalized the white blood cell count in the tumor-bearing animals, indicating its potential immuno-stimulatory effect. Aim of the studyTo evaluate the immune stimulatory effects of F3 from S. crispus in NMU-induced rat mammary tumor model. Materials and methodsImmunohistochemistry analysis of cellular immune parameters (CD4+ or CD8+ T cells, CIITA, MHC-II and CD68) was performed on NMU-induced rat mammary tumor nodules, followed by evaluation of the serum level of 34 cytokines using the cytokine antibody array. ResultsSignificant increase in MHC-II, CD4+ and CD8+ T cell and CIITA expression by tumor cells was observed in F3-treated rats compared to the tumor control group. F3-treated rats also displayed a significant decrease in the serum level of CCL2 and CD68+ infiltrating macrophages. Serum IFN-γ level in this group was increased by 1.7-fold suggesting enhanced infiltration of T cells, and upregulation of CIITA and MHC-II expression in the tumor cells might be triggered by F3-induced production of IFN-γ. ConclusionOur findings demonstrated for the first time that a subfraction from S. crispus, F3, is capable of activating the immune system in rats-bearing NMU-induced mammary tumor, which may contribute to the anticancer effects of F3, and additionally support the traditional use of S. crispus leaves to boost the immune system.

Blood flow, volume and arterio-venous passages in induced mammary tumours of the rat

ObjectiveTo study blood flow, vascular volume and arterio-venous passages in induced mammary tumours of the rat to characterize parameters possibly responsible for tumour hyponutrition. MethodDimethylbenzanthracene-induced mammary tumours in Sprague-Dawley rats were studied. Regional blood flow was studied by use of the radioactive microsphere tracer technique using 141Cerium-labelled 15μm spheres coinjected into the left cardiac ventricle with 125Iodine-labelled 25μm spheres. Blood volume was studied by use of 125Iodine- or 99mTechnetium-labelled human serum albumin, the latter allowing autoradiography of tumour sections for visualization of flow and volume. ResultsTwenty-seven rats with 170 tumours had a mean tumour blood flow of 48 and 67mL×min−1×100g−1 using 15 and 25μm sphere data, respectively, indicating a significant passage through vessels between 15 and 25μm. The lungs showed a “nominal bronchial” blood flow of 260 and 135mL×min−1×100g−1 for the 15 and 25μm spheres, respectively, indicating pulmonary trapping, particularly of small spheres passing the systemic circulation in vessels larger than 15μm. There was a positive correlation between the total tumour blood flow within individual rats and trapped spheres of both dimensions in the lungs, indicating shunts also larger than 25μm.Normal tissues disclosed only small differences in regional blood flow as measured by the two spheres.Blood volume was studied in 20 rats with 120 tumours, with a vascular volume of 3.6mL×100g−1 representing a blood turnover >15 times/min. Blood volume co-localized with perfusion as seen in autoradiographs. ConclusionIn induced rat mammary tumours, a high fraction of blood, 28%, passes arterio-venous vessels between 15 and 25μm and there also exist passages >25μm. These findings indicate that the functional capacity of the tumour vascular bed might be impaired, adding to the abnormal microenvironment of tumours.

Dampness-Heat Accelerates DMBA-Induced Mammary Tumors in Rats.

To investigate the impact of dampness-heat (DH) on the development of mammary tumors in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rats. Forty rats were randomly divided into 3 groups in a randomized block design, including the control group (n=13), DMBA group (n=14), and DMBA plus DH group (n=13). Rats in the DMBA group and DMBA plus DH group were intragastrically administrated with DMBA (100 mg/kg) for twice, once per week, while rats in the control group were treated with equivalent volumes of sesame oil. After DMBA administration, rats in the DMBA plus DH group were exposed to a simulated climate chamber with ambient temperature (33.0±0.5°C) and humidity (90%±5%) for 8 weeks, 8 h per day. The body weight, time of tumor formation, and number of tumors were measured weekly to calculate tumor incidence, average latency period, average number of tumors, and average tumor weight. At the end of the experiment, the levels of matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinases 1 (TIMP-1) in serum, and the contents of tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β in serum and tumor tissue were measured, respectively. Some tumor tissues were processed for hematoxylin-eosin staining to determine the histopathological changes. Compared with DMBA, DMBA plus DH significantly increased the average number of tumors, average tumor weight, levels of serum MMP-9, TIMP-1, TNF-α and IL-1β, and contents of tumor tissue TNF-α and IL-1β (P<0.05 or P<0.01). DH could accelerate the development of mammary tumors through increasing the expressions of MMP-9, TIMP-1, TNF-α and IL-1β in DMBA-induced rats.

Determination of temperature distribution in tissue for interstitial cancer photothermal therapy

Background: Temperature increase in tumour tissue during photothermal therapy (PTT) is a significant factor in determining the outcomes of the treatment. Therefore, controlling and optimising temperature distribution in target tissue is crucial for PTT. In this study, we developed a unique ex vivo device to study the temperature distribution during PTT to be used as a guide for the desired photothermal effects for cancer treatment.Methods: Bovine liver tissue buried inside agarose gel served as a phantom tumour surrounded by normal tissue. A thermostatic incubator was used to simulate tissue environment in live animals. The temperature distributions were measured by thermocouples with needle probes at different locations inside the target tissue, during laser irradiation using an 805-nm laser.Results: The results obtained using the ex vivo device were verified by comparing the tissue temperature directly measured in animal tumours irradiated under the same conditions. With this model, the spatial distribution of temperature in target tissue can be monitored in real time. A two-dimensional temperature distribution in target tissue allows us to establish the correlations among laser parameters, temperature distribution and tumour size. In addition, the optimal temperature range for tumour destruction and immunological stimulation was determined using metastatic rat mammary tumour model.Conclusion: The device and method developed in this study can provide guidance for choosing the appropriate treatment parameters for optimal photothermal effects, particularly when combined with immunotherapy, for cancer treatment.

Selective effects of whey protein concentrate on glutathione levels and apoptosis in rats with mammary tumors

Glutathione (GSH) plays an important role in antioxidant defense and regulation of apoptosis. GSH deficiency is related to many diseases, including cancer, and increased GSH levels in cancer cells are associated with chemotherapy resistance because of resistance to apoptosis. In this study, we investigated the effects of whey protein concentrate (WPC), a precursor of GSH, in rats with mammary tumors induced by treatment with 7,12-dimethylbenz(a)anthracene (DMBA). DMBA treatment results in cellular changes that mimic the initiation and promotion of carcinogenesis of breast tissue. We aimed to examine the possible preventive effects of diets containing whey protein on DMBA-induced mammary tumors in rats. The results indicate that WPC (0.334 g/kg) supplementation significantly increased the liver GSH levels by 92%, and were accompanied by low Bax/Bcl-2 ratio (from 5 to 3) and cleaved caspase-3/procaspase-3 ratio (from 2.4 to 1.2) in DMBA-treated rats. Furthermore, tumor GSH levels were decreased by 47% in WPC-supplemented rats, which resulted in increased Bax/Bcl-2 ratio (from 0.9 to 2) and cleaved caspase-3/procaspase-3 ratio (from 1.1 to 2.7). In conclusion, supplementation with WPC could selectively deplete tumor GSH levels and, therefore, WPC supplementation might be a promising strategy to overcome treatment resistance in cancer therapy.

Abstract 1798: Mortalin precursor as potential marker for chemoprevention with SHetA2

Abstract Introduction: The flexible heteroarotinoid (Flex-Het), SHetA2, is a novel anticancer drug that induces both intrinsic and extrinsic apoptosis and autophagy in cancer cells, but not in normal cells. Protein isolation / mass spectrometry analysis using SHetA2-coupled polystyrene magnetic beads yielded three SHetA2-binding proteins all belonging to HSPA family namely, HSPA9/mortalin, HSPA8/Hsc70 and HSPA5/BiP. Mortalin, in addition to its vital chaperoning roles in other organelles of the cell, is essential for import and folding of nuclear-encoded mitochondrial proteins. The precursor form of mortalin has a 46-amino acid N-terminal region that functions as a mitochondrial localization sequence (MLS) and is cleaved by proteases after import into the mitochondrial matrix. Hypothesis: SHetA2 binding to mortalin causes alterations that can be measured to study mechanism and monitor drug effects in animal models and clinical trials. Methods: Western blots of whole cell or subcellular protein extracts made from cultures of normal human fallopian tube secretory or mammary epithelial cells, rat mammary tumors or human ovarian cancer cell lines treated with SHetA2 or solvent were probed with an antibody that recognizes total mortalin, a custom-made rabbit polyclonal antibody specific for the mortalin MLS (PA8238) or antibodies to loading control proteins. Immunocytochemistry using these antibodies was performed with an automated (Leica Bond III) IHC work station on cells treated with SHetA2 or solvent. Results: Using a commercial antibody against total mortalin, we observed a lower-mobility band closely moving above the specific (mature) mortalin band in Western blots. Our mortalin MLS-specific antibody recognized only the lower-mobility band confirming it as the precursor form of mortalin in the SHetA2-treated cell extracts. Subcellular fractionation of the drug-treated cells revealed that the precursor protein accumulated only in the cytoplasm and not in the mitochondria. Staining of whole cells with an antibody to total mortalin showed no effect of SHetA2 treatment on the punctate pattern of expression consistent with mitochondrial localization. In contrast, staining with the mortalin MLS-specific antibody demonstrated that SHetA2 increased the intensity of the diffuse cytoplasmic staining consistent with localization of mortalin throughout the cytoplasm. These results were consistently observed among the various cell types. Conclusions: SHetA2 is the only known drug that blocks the translocation of the precursor mortalin to mitochondria and its processing to mature protein. Such cytoplasmic accumulation of the precursor form of mortalin can potentially serve as pharmacodynamic endpoint to study SHetA2’s effect in laboratory experimental tumor models as well as in human clinical trials. Funding: NCI PREVENT Task Order HHSN26100002 Citation Format: Elangovan Thavathiru, Vishal Chandra, Rajani Rai, Doris Benbrook. Mortalin precursor as potential marker for chemoprevention with SHetA2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1798. doi:10.1158/1538-7445.AM2017-1798

A Contrast-Enhanced Ultrasonographic Study About the Impact of Long-term Exercise Training on Mammary Tumor Vascularization.

This study aimed to evaluate the impact of long-term exercise training on the vascularization of rat mammary tumors. Female rats were divided into 4 groups: N-methyl-N-nitrosourea (MNU) treated sedentary, MNU treated exercised, control sedentary, and control exercised. Tumor development was induced in the MNU groups by MNU administration. Exercised groups were trained for 35 weeks. Tumor vascularization was evaluated by pulsed Doppler and contrast-enhanced ultrasonography. The pulsatility and resistive indices were slightly higher in the MNU sedentary group (P > .05). Mammary tumors mainly had centripetal and heterogeneous enhancement of the contrast, clear margins, and the presence of penetrating vessels. The MNU exercised group had a lower arrival time and time to peak and higher peak intensity, wash-in, and wash-out (P > .05). The area under the curve was similar between groups (P > .05). The contrast-enhanced ultrasonographic study did not detect differences in mammary tumor vascularization between MNU sedentary and MNU exercised groups previously detected by power Doppler imaging, B-flow imaging, and immunohistochemistry.

Prognostic factors in MNU and DMBA-induced mammary tumors in female rats

Chemically-induced mammary tumors in rats by the carcinogens 1-methyl-1-nitrosourea- (MNU) and 7,12-dimethylbenz[a]anthracene (DMBA) are the most widely used models for studies related with human breast cancer. This study aimed to evaluate the immunoexpression of the prognostic factors estrogen receptor α (ERα), progesterone receptor (PR) and Ki-67, in MNU and DMBA-induced rat mammary tumors, in order to know the model that best suits to woman breast cancer. Twenty-eight MNU-induced and 16 DMBA-induced mammary tumors in virgin female Sprague-Dawley rats were analyzed. The expression of the prognostic markers ERα, PR and Ki-67 proliferation index (Ki-67 PI) was assessed by immunohistochemistry. Mitotic activity index (MAI) was also evaluated. More than one histological pattern was identified in each mammary tumor. Carcinomas constituted the lesions most frequently induced by both carcinogens: 33 MNU-induced carcinomas and 23 DMBA-induced carcinomas. All MNU and DMBA-induced mammary carcinomas were ER+/PR+, with a higher expression of ERα when compared with PR. Tumors’ weight, the expression of ERα, PR, Ki-67 PI and MAI were higher in MNU-induced mammary carcinomas when compared with the DMBA-induced ones. Statistically significant differences between groups were observed for ERα, PR and MAI (p<0.05). The higher KI-67 PI and MAI in MNU-induced mammary carcinomas are suggestive of a higher aggressiveness of these carcinomas when compared with the DMBA-induced ones, and consequently a worse response to the therapy and a worse prognosis. In this way, the use of the rat model of MNU-induced mammary tumors is advised in experimental protocols aiming to study more aggressive mammary tumors within the group of double-positive mammary tumors (ER+/PR+).

Hexachlorobenzene alters cell cycle by regulating p27-cyclin E-CDK2 and c-Src-p27 protein complexes

Hexachlorobenzene (HCB) is an organochlorine pollutant widely distributed in the environment around the entire world. Previous reports from our group and others have demonstrated that this compound is as an endocrine disruptor. We have also reported that HCB presents a co-carcinogenic effect in N-Nitroso-N-methyl-urea–induced mammary tumours in rats. In this work, we studied the effects of HCB on cell cycle progression and cell cycle regulating protein expression in the estrogen-sensitive breast cancer cell line, MCF-7. Here, we show that HCB alters cell cycle in a concentration-dependent way. The lowest assessed concentration (0.005μM) promotes the cell cycle progression, enhances cyclin D1 expression, and reduces the nuclear localization of p27 accompanied by an increased interaction between p27 and c-Src kinase. On the other hand, 5μM HCB delays the cell cycle progression and promotes the formation of the cyclin E-CDK2-p27 protein complex. Our results show that HCB stimulates cell proliferation through cell cycle modulation and c-Src involvement in MCF-7 cells. Here, we report for the first time that differential mechanisms of action of HCB on mammary cell cycle progression are triggered at different concentrations of this pollutant.

Inhibition of Mammary Tumourigenesis in the MNU-Induced Rat Mammary Tumour Model by Propolis

Inhibition of Mammary Tumourigenesis in the MNU-Induced Rat Mammary Tumour Model by Propolis

New orally active DNA minor groove binding small molecule CT-1 acts against breast cancer by targeting tumor DNA damage leading to p53-dependent apoptosis.

Targeting tumor DNA damage and p53 pathway is a clinically established strategy in the development of cancer chemotherapeutics. Majority of anti-cancer drugs are delivered through parenteral route for reasons like severe toxicity, lack of stability, and poor enteral absorption. Current DNA targeting drugs in clinical like anthracycline suffers from major drawbacks like cardiotoxicity. Here, we report identification of a new orally active small molecule curcumin-triazole conjugate (CT-1) with significant anti-breast cancer activity in vitro and in vivo. CT-1 selectively and significantly inhibits viability of breast cancer cell lines; retards cells cycle progression at S phase and induce mitochondrial-mediated cell apoptosis. CT-1 selectively binds to minor groove of DNA and induces DNA damage leading to increase in p53 along with decrease in its ubiquitination. Inhibition of p53 with pharmacological inhibitor as well as siRNA revealed the necessity of p53 in CT-1-mediated anti-cancer effects in breast cancer cells. Studies using several other intact p53 and deficient p53 cancer cell lines further confirmed necessity of p53 in CT-1-mediated anti-cancer response. Pharmacological inhibition of pan-caspase showed CT-1 induces caspase-dependent cell death in breast cancer cells. Most interestingly, oral administration of CT-1 induces significant inhibition of tumor growth in LA-7 syngeneic orthotropic rat mammary tumor model. CT-1 treated mammary tumor shows enhancement in DNA damage, p53 upregulation, and apoptosis. Collectively, CT-1 exhibits potent anti-cancer effect both in vitro and in vivo and could serve as a safe orally active lead for anti-cancer drug development. © 2016 Wiley Periodicals, Inc.

Lipid mapping by desorption electrospray ionization mass spectrometry in a murine breast DMBA carcinogenesis model

Lipids are molecules with a large structural diversity inserted in various cytological and pathological processes, such as in breast cancer. The knowledge of the metabolic and lipid profiles in breast cancer helps to identify and classify neoplastic and non-neoplastic areas and aids therefore in precise histological diagnosis and in the identification of new promising therapeutic targets. The present study aimed to compare non-neoplastic samples of Sprague-Dawley rat breast lesions to a DMBA chemical carcinogenesis model via DESI-MS imaging. The model Sprague-Dawley rats were treated with the carcinogen compound DMBA, and frozen mammary samples with or without tumors were imaged. The developed ductal carcinomas showed indeed contrasting lipid relative abundances as compared to non-neoplastic breast tissue. Most lipids belong to the fatty acyl family, and were detected in greater abundance in the non-neoplastic cancer samples. The lipid ion of m/z 336 was observed only in the neoplastic areas. This is the first study that demonstrates changes in lipid profile of mammary tumors in rats treated with DMBA, and the promising results seems to encourage and serve as a guide for further lipidomic studies regarding breast cancer.

Abstract 2860: Loss of expression of the metastasis suppressor CREB3L1 is associated with high-grade metastatic breast cancer and poorer prognosis

Abstract The unfolded protein response (UPR) is required for proper protein folding in the endoplasmic reticulum under conditions of stress such as those encountered in the tumor microenvironment. Recently, a number of studies have suggested a role for the unfolded protein response in the development of cancer, more specifically in regulating the balance between cell death, dormancy and the growth of cancer cells in the tumor microenvironment. This study examined the role of CREB3L1 (cAMP-responsive element-binding protein 3-like protein 1), a member of the UPR, in breast cancer development and metastasis. Initial experiments identified the loss of CREB3L1 expression in metastatic breast cancer cell lines compared to low- or non-metastatic cell lines. When metastatic cells were transfected with CREB3L1 they demonstrated reduced invasion and migration in vitro, as well as a significantly decreased ability to survive under non-adherent or hypoxic conditions. Interestingly, in an in vivo rat mammary tumor model, CREB3L1 expressing cells not only failed to form metastases compared to CREB3L1 null cells but regression of the primary tumors was seen in 70% of the animals as a result of impaired angiogenesis. Microarray and ChIP on Chip analyses identified changes in the expression of many genes involved in cancer development and metastasis, including a decrease in those involved in angiogenesis. To determine if these findings translated to human breast cancer, real-time PCR analysis of CREB3L1 expression in human breast cancer tissues of differing grade (n = 213) was performed. Expression of CREB3L1 was elevated in low and medium grade tissue but substantially reduced in high grade tissue, compared to normal. Analysis of the on-line TCGA database of breast cancer samples identified a strong inverse relationship between CREB3L1 gene methylation and the loss of CREB3L1 mRNA expression. Low expression of CREB3L1 was associated with a poorer prognosis with a shorter relapse-free survival for luminal A and triple negative breast cancer patients. These data suggest that CREB3L1 plays an important role in suppressing tumorgenesis and loss of expression is required for the development of a metastatic phenotype. In addition CREB3L1 expression may be a useful marker for determining patient prognosis. Citation Format: Paul Mellor, Alison Ward, Deborah Anderson. Loss of expression of the metastasis suppressor CREB3L1 is associated with high-grade metastatic breast cancer and poorer prognosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2860.

Molecular Profiling and Malignant Behavior Define Two Rat Mammary Tumor Cell Lines as a Relevant Experimental Model

Cancer cell lines have become a reliable tool in genetic and biochemical studies of breast cancer. Here, we described the behavior and novel molecular characterization of two cell lines derived from DMBA-induced rat mammary tumor, LA7 and RBA. LA7 cells have been identified as myoepithelial cells with stem cell properties, whereas the RBA cell line are epithelial cells that present mutational activated H-Ras, but are much less known. We evaluated the proliferation rate and molecular markers, several signaling pathways status related to proliferation, survival, inflammation, and apoptosis, as well as migration capacity, global DNA methylation levels, and stem cells populations. In fact, we found the A/T transversion in the c-Ha-Ras codon 61 as the activator mutation origin described in RBA cells. LA7 and RBA cells showed a high proliferation rate associated with overexpression of Cyclin D1, and resistance to apoptotic signals due to lack of expression of Bad. Moreover, neither of these two cell lines expressed steroid receptors, but they showed high migration capacity, all in accordance with an aggressive phenotype. We found global DNA methylation levels in LA7 and RBA cells lower than reference tissues analyzed, in addition to the presence of different stem cells populations in RBA cell line that differed in the expression of CD44 and CD24. These results revealed a malignant behavior associated with cancer stem cell phenotype. Since this profile is similar to a human triple-negative basal-like tumor, their extensive characterization presented herein increases their value as a good in vitro model. J. Cell. Biochem. 117: 2825-2834, 2016. © 2016 Wiley Periodicals, Inc.

Abstract P3-11-01: Telapristone acetate suppresses the expression of genes involved in cell proliferation, stem cell self-renewal, and anti-apoptosis in MNU-induced rat mammary tumors

Abstract Background: We previously reported that telapristone acetate (TPA), an anti-progestin, significantly suppressed mammary tumor formation and growth accelerated by progesterone (P4) and medroxyprogesterone acetate (MPA) in N-methyl-N-nitrosourea (MNU)-induced carcinogenesis model[1]. This suggests TPA holds promise for the prevention and treatment of human breast cancer. The purpose of this study is to identify the genes modulated by TPA in rat mammary ER/PR+ tumors, which may be translated into a therapeutic biomarker of TPA therapy in ER/PR+ breast cancer. Methods: Rat mammary tumors from MNU carcinogenesis were used for study [1]. Treatment groups were: control, MPA, P4, MPA+TPA, and P4+TPA. Doses were 30mg of TPA and 25mg of P4 or MPA (90 day release pellets). Tumor epithelium was isolated by laser capture microdissection and RNA extracted. 100 ng of RNA per sample was used for targeted -gene expression profiling by nCounter Gene Expression analysis (Nanostring Inc.). 112 genes related to PR, GR, AR signaling were selected by MetaCore pathway analysis (Thomson Reuters, Inc.). The data was normalized by positive control and RNA content using nSolver. The genes significantly modulated by hormones and TPA were reported as means ± standard deviations (SD) for each group as well as a p-value for pairwise t-test comparison. Results: mRNA expression, log mean (log SD).Control vs. P4GeneControlP4pCHD51.5 (0.6)0.7 (0.5)0.03P4 vs. P4+TPAGeneP4P4+TPApSurvivin0.7 (0.5)3.9 (0.2)0.02KI677.6 (0.3)7.1 (0.3)0.02FANCA4.3 (0.2)3.8 (0.3)0.03EZH25.4 (0.2)5.1 (0.2)0.03RFC35.6 (0.2)5.3 (0.2)0.03C.MYC5.9 (0.4)5.5 (0.3)0.04TK15.2 (0.3)4.8 (0.2)0.05Control vs. MPAGeneControlMPApFKBP54.7 (0.4)5.6 (0.6)0.00TNFSF112.0 (0.2)2.8 (0.9)0.02TEK3.7 (0.5)3.1 (0.5)0.03PLZF2.8 (0.9)4.0 (1.1)0.03EPAS15.5 (0.5)4.9 (0.4)0.04MPA vs. MPA+TPAGeneMPAMPA+TPApEGFR6.4 (0.2)6.0 (0.4)0.02SNAI25.0 (0.3)4.6 (0.4)0.04AR4.2 (0.8)5.0 (0.7)0.04Endothelin-15.0 (0.7)3.1 (0.6)0.04 Compared to the control, P4 significantly reduced CHD5 expression, a tumor suppressor in a mouse model, and its down-regulation contributes to the development and progression of human breast cancer. Comparing the P4 and P4+TPA groups, the genes involved in cell proliferation and DNA repair (C-MYC, FANCA, Ki67, RFC3, and TK1), EZH2 (stem cell self-renewal), and anti-apoptosis (survivin) were significantly down-regulated by TPA. On the other hand, MPA induced the up-regulation of FKBP5 (androgen-regulated protein), PLZF (GR responsive gene), TEK (angiogenesis-regulating gene) and TNFSF11 (RANKL) compared to the control. Comparing the MPA and MPA+TPA groups, TPA significantly reduced the expression of EGFR, SNAI2 and endothelin-1 (EMT initiation or maintenance) and increased AR expression. Conclusions: TPA inhibits P4/PR mediated cell proliferation, and increases apoptosis. The tumorigenic effect of MPA is likely through AR and GR rather than a PR specific manner. In addition to the PR, MPA binds with high affinity to AR and GR, and therefore has the potential to elicit actions distinct to those of P4, which does not bind AR. [1] Lee O, et al. Cancer Res 2013 vol. 73 no. 24 Supplement P4-11-02. Citation Format: Lee O, Clare SE, Ivancic D, Scholtens DM, Khan SA. Telapristone acetate suppresses the expression of genes involved in cell proliferation, stem cell self-renewal, and anti-apoptosis in MNU-induced rat mammary tumors. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-11-01.

NMR-Based Lipidomic Approach To Evaluate Controlled Dietary Intake of Lipids in Adipose Tissue of a Rat Mammary Tumor Model.

The fatty acids composition of adipose tissue may provide information on the nutritional part of the risk or evolution of breast cancer. To determine whether (1)H NMR of adipose tissue provides information on the nature of the diet consumed, a dietary intervention with increasing percentage of polyunsaturated n-3 docosahexaenoic acid (DHA 22:6n-3, provided as DHASCO oil) was applied to a rat model of N-nitroso-N-methylurea-induced mammary tumors. Spectra of the lipid extracts were obtained from adipose tissues in five groups of Sprague-Dawley rats fed with a diet containing 7% peanut/rapeseed enriched with 8% (w/w) of an oil without (palm oil) or with low (1%), moderate (3%), or high (8%) DHASCO content. A control group received a basal diet with 15% peanut/rapeseed representative of the "Western" diet. After 5 months of those five controlled diets, adipose tissue was collected for analysis of the lipid extract using both (1)H NMR analysis on an 11.7 T spectrometer and gas chromatography considered as gold standard. (1)H NMR analysis showed a dose-dependent increase in DHA in the lipid extract of adipose tissues and a commensurate decrease in n-6 polyunsaturated fatty acids in the three DHA groups, which allowed one to follow n-6/n-3 ratio changes. The highest n-6/n-3 ratio was observed in the control Western diet group compared to the other diet groups. The integrated spectral regions showed separation between groups, thereby documenting a specific NMR lipid profile corresponding to each dietary intervention. Those diet-dependent NMR lipid profiles were consistent with that obtained with gas chromatography analyses of the same samples. This study is a proof of concept highlighting the potential use of the (1)H NMR approach to evaluate dietary intervention in biopsies of adipose tissues.

Long chain n-3 polyunsaturated fatty acids increase the efficacy of docetaxel in mammary cancer cells by downregulating Akt and PKCε/δ-induced ERK pathways

Taxanes can induce drug resistance by increasing signaling pathways such as PI3K/Akt and ERK, which promote survival and cell growth in human cancer cells. We have previously shown that long chain n-3 polyunsaturated fatty acids, such as docosahexaenoic acid (DHA, 22:6n-3) decrease resistance of experimental mammary tumors to anticancer drugs. Our objective was to determine whether DHA could increase tumor sensitivity to docetaxel by down-regulating these survival pathways. In docetaxel-treated MDA-MB-231 cells, phosphorylated-ERK1/2 levels were increased by 60% in membrane and nuclear compartments, compared to untreated cells. Our data showed that ERK1/2 activation depended on PKC activation since: i) enzastaurin (a pan-PKC inhibitor) blocked docetaxel-induced ERK1/2 phosphorylation ii) docetaxel increased PKC activity by 30% and phosphatidic acid level by 1.6-fold iii) inhibition of PKCε and PKCδ by siRNA resulted in reduced phosphorylated ERK1/2 levels. In DHA-supplemented cells, docetaxel was unable to increase PKCε and δ levels in membrane and nuclear fractions, resulting in diminished ERK1/2 phosphorylation and increased docetaxel efficacy. Reduced membrane level of PKCε and PKCδ was associated with significant incorporation of DHA in all phospholipids, including phosphatidylcholine which is a major source of phosphatidic acid. Additionally, examination of the Akt pathway showed that DHA could repress docetaxel-induced Ser473Akt phosphorylation. In rat mammary tumors, dietary DHA supplementation during docetaxel chemotherapy repressed ERK and Akt survival pathways and in turn strongly improved taxane efficacy. The P-ERK level was negatively correlated with tumor regression. These findings are of potential clinical importance in treating chemotherapy-refractory cancer.

Quantitative Ultrasound for Monitoring High-Intensity Focused Ultrasound Treatment In Vivo.

The success of any minimally invasive treatment procedure can be enhanced significantly if combined with a robust noninvasive imaging modality that can monitor therapy in real time. Quantitative ultrasound (QUS) imaging has been widely investigated for monitoring various treatment responses such as chemotherapy, radiation, and thermal therapy. Previously, we demonstrated the feasibility of using spectral-based QUS parameters to monitor high-intensity focused ultrasound (HIFU) treatment of in situ tumors in euthanized rats [Ultrasonic Imaging 36(4), 239-255, 2014]. In the present study, we examined the use of spectral-based QUS parameters to monitor HIFU treatment of in vivo rat mammary adenocarcinoma tumors (MAT) where significant tissue motion was present. HIFU was applied to tumors in rats using a single-element transducer. During the off part of the HIFU duty cycle, ultrasound backscatter was recorded from the tumors using a linear array co-aligned with the HIFU focus. A total of 10 rats were treated with HIFU in this study with an additional sham-treated rat. Spectral parameters from the backscatter coefficient, i.e., effective scatterer diameter (ESD) and effective acoustic concentration (EAC), were estimated. The changes of each parameter during treatment were compared with a temperature profile recorded by a fine-needle thermocouple inserted into the tumor a few millimeters behind the focus of the HIFU transducer. The mean ESD changed from 121 ±6 to [Formula: see text], and the EAC changed from 33 ±2 to [Formula: see text] during HIFU exposure as the temperature increased on average from 38.7 ±1.0 (°)C to 64.2 ±2.7 (°)C. The changes in ESD and EAC were linearly correlated with the changes in tissue temperature during the treatment. When HIFU was turned off, the ESD increased from 81 ±8 to [Formula: see text] and the EAC dropped from 46 ±3 to 36±2 dB/cm(3) as the temperature decreased from 64.2 ±2.7 (°)C to 45 ±2.7 (°)C. QUS was demonstrated in vivo to track temperature elevations caused by HIFU exposure.