Patent Application
20070207975
The invention relates to the field of medicine and, more particularly, to the field of cancer medicine.
Epidemiological studies indicate that women in countries with high-fat diets have a risk of breast cancer that can be five-fold higher than that of women in countries with low-fat consumption [1-3], strongly suggesting that a high intake of dietary fat could increase breast cancer risk [3]. This dietary fat hypothesis has been supported by a number of epidemiological, experimental and mechanistic data, collectively providing evidence that dietary or exogenously provided fats may play a role in the carcinogenesis, evolution and/or progression of breast cancer [3-5]. However, case-control, cohort and recent prospective epidemiological studies have generated conflicting results, and taken together do not support a strong association [6-9].
Research in experimental animals has yielded inconsistent results, having attributed a range of effects of dietary fat extending from a non-promoting or a low-promoting effect to a protective one on breast cancer [1, 14-16]. These conflicting results may be explained in part by the fact that olive oil is administered as a mixture of oil containing several fatty acids and glycerol, as well as natural chemoprotectants (tocopherols, carotenoids, polyphenols, and the like) [17-19], e.g., antioxidants in the unsaponifiable fraction of the oil.
The Her-2/neu oncogene (also called neu and erbB-2) represents one of the most important oncogenes in breast cancer. Her-2/neu codes for the p185Her-2/neu oncoprotein, a transmembrane tyrosine kinase orphan receptor [22, 23]. Her-2/neu amplification and overexpression occurs in 20% of breast carcinomas and is correlated with unfavorable clinical outcome [24-26]. Expression of high levels of Her-2/neu is sufficient to induce the neoplastic transformation of some cell lines [27, 28], suggesting a role for Her-2/neu in the etiology of some breast carcinomas. Indeed, Her-2/neu is overexpressesed not only in invasive breast cancer, but also in pre-neoplastic breast lesions, such as atypical duct proliferations and in ductal carcinoma of the breast in situ [29-31]. Moreover, Her-2/neu is a metastasis-promoting gene, enhancing the invasive and metastatic phenotype of breast cancer cells [32, 33]. Her-2/neu overexpression is also associated with resistance to chemo- and endocrine therapies [34, 35], while representing a successful therapeutic target of the biotechnology era, as exemplified by the drug trastuzumab (Herceptin®; Genentech, San Francisco, Calif.). Trastuzumab is a humanized monoclonal IgG1, binding with high affinity to the ectodomain of p185Her-2/neu that has clinical activity in a subset of breast cancer patients, thus confirming the role of Her-2/neu in the progression of some breast carcinomas [36-39].
Although data suggest that trastuzumab may be useful in select cases of advanced breast cancer, these benefits are modest and usually do not represent a cure. Moreover, not all Her-2/neu-overexpressing respond to treatment with trastuzumab and its clinical benefit is limited by the fact that resistance develops rapidly in virtually all treated patients [40]. Although the molecular mechanisms underlying trastuzumab resistance have begun to emerge [41-43], there are no data concerning strategies able to sensitize breast cancer cells to the growth-inhibitory activity of anti-p185Her-2/neu antibodies, such as trastuzumab.
Thus, a need continues to exist in the art for effective, and preferably safe, approaches to the treatment of a variety of cancers, including breast carcinomas, in man and other animals. This need is so pronounced that single-therapeutic, as well as combination therapies, are desperately being sought.
The invention provides materials and methods useful in treating a variety of cancers, as well as preventing such cancers and ameliorating at least one symptom associated with such cancers, by administering a therapeutically or prophylactically effective amount of an unsaturated C16-C22 trans-fatty acid. The cancers amenable to the methods of the invention overexpress both p185Her-2/neu, the erbB-2 (neu) gene product, and fatty acid synthase relative to non-cancerous cells of the same type. The materials and methods of the invention are suitable for use in combination with known anti-cancer therapeutics and therapies, particularly those that reduce the activity of p185Her-2/neu. An exemplary known anti-cancer therapeutic contemplated for combination with the materials and/or methods of the invention is an antibody specifically recognizing p185Her-2/neu, such as trastuzumab.
In one aspect, the invention provides a method of treating a cancer cell overexpressing p185Her-2/neu and fatty acid synthase comprising administering a therapeutically effective amount of an unsaturated trans-fatty acid to an organism comprising the cancer cell. In some embodiments, a nucleic acid overexpressing p185Her-2/neu comprises a promoter comprising a PEA3 binding site. This aspect of the invention comprehends methods wherein the organism in need is a human. An exemplary cancer amenable to treatment by the method include a cancer selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, colorectal cancer, bladder cancer, stomach cancer, lung cancer, oral cancer of the tongue and cancer of the endometrium. Consistent with the statement above, the method embraces embodiments wherein the fatty acid is selected from the group consisting of C16-C22 fatty acids. Preferably, the fatty acid is a naturally occurring mono- or polyunsaturated trans-fatty acid. In particular, embodiments of the method are contemplated wherein the fatty acid is selected from the group consisting of an omega-9 unsaturated fatty acid, an omega-6 unsaturated fatty acid and an omega-3 unsaturated fatty acid. Exemplary fatty acids suitable for use in the method include a fatty acid selected from the group consisting of oleic acid, γ-linolenic acid and α-linolenic acid.
Another aspect of the invention is drawn to a method of treating a cancer cell overexpressing p185Her-2/neu and fatty acid synthase comprising (a) administering a first anti-cancer therapeutic to an organism comprising the cancer cell, wherein the first anti-cancer therapeutic reduces the activity of p185Her-2/neu; and (b) delivering a fatty acid according to the above-described method to the organism. In preferred embodiments of this aspect of the invention, the anti-cancer effect of the combined treatment is greater than the additive effect of two separate treatments (i.e., the effect is a synergistic effect). This aspect of the invention also comprehends embodiments of the method wherein the organism is a human. Some embodiments of this aspect of the invention comprise a nucleic acid overexpressing p185Her-2/neu, wherein the nucleic acid comprises a promoter comprising a PEA3 binding site. Cancers amenable to treatment by the method include a cancer selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, colorectal cancer, bladder cancer, stomach cancer, lung cancer, oral cancer of the tongue and cancer of the endometrium. Preferred fatty acids for use in this aspect of the invention are unsaturated C16-C22 trans-fatty acids, such as an omega-9 unsaturated fatty acid , an omega-6 unsaturated fatty acid and an omega-3 unsaturated fatty acid, as exemplified by oleic acid, γ-linolenic acid and α-linolenic acid. Any known anti-cancer therapeutic may be used as the first anti-cancer therapeutic, provided it contributes to a lowering of the activity level of p185Her-2/neu. Preferred anti-cancer therapeutics for use as the first anti-cancer therapeutic in the method of the invention are antibodies that specifically recognize p185Her-2/neu, such as trastuzumab.
Another aspect of the invention is drawn to a method of reducing the risk of developing a cancer comprising a cell over-expressing p185Her-2/neu and fatty acid synthase, the method comprising delivering a prophylactically effective amount of a fatty acid as described above. In some embodiments, the method comprises a fatty acid that is an unsaturated C16-C22 trans-fatty acid, such as an omega-9 unsaturated fatty acid, an omega-6 unsaturated fatty acid or an omega-3 unsaturated fatty acid, as exemplified by oleic acid, γ-linolenic acid and α-linolenic acid.
Yet another aspect of the invention is drawn to a kit for treatment of a cancer cell overexpressing p185Her-2/neu and fatty acid synthase comprising a compound that specifically inhibits the binding activity of p185Her-2/neu, a fatty acid and a protocol for the treatment. In some embodiments, the compound that specifically inhibits the binding activity of p185Her-2/neu is an antibody that specifically binds p185Her-2/neu, such as trastuzumab. In some embodiments, the kit comprises a fatty acid that is an unsaturated C16-C22 trans-fatty acid, such as an omega-9 unsaturated fatty acid, an omega-6 unsaturated fatty acid or an omega-3 unsaturated fatty acid, as exemplified by oleic acid, γ-linolenic acid and α-linolenic acid.
Numerous other aspects and advantages of the present invention will be apparent upon consideration of the drawing and detailed description.
The present invention provides materials and methods useful alone or in combination with therapeutics/therapies in the treatment or prevention of a variety of cancers characterized by cancer cells overexpressing p185Her-2/neu (i.e., Her-2/neu) and exhibiting elevated levels of fatty acid synthase (i.e., FAS). The materials of the invention are kits comprising fatty acids, such as purified forms of naturally occurring unsaturated C16-C22 trans-fatty acids. Preferred forms of the fatty acids include monounsaturated ω-9 fatty acids (e.g., oleic acid), polyunsaturated ω-6 fatty acids (e.g., γ-linolenic acid), and polyunsaturated ω-3 fatty acids (e.g., α-linolenic acid). Suitable cancers include any cancer characterized by cancer cells overexpressing p185Her-2/neu and having elevated FAS activity; including breast cancer, ovarian cancer, prostate cancer, colorectal cancer, bladder cancer, stomach cancer, lung cancer, oral cancer of the tongue and cancer of the endometrium.
The invention comprehends use of the materials of the invention alone or in combination with any known anti-cancer treatment. By way of exemplifying a combined treatment method, a therapeutically effective amount of a fatty acid according to the invention is administered before, after, or concomitantly with, an antibody specifically recognizing or binding to p185Her-2/neu, such as trastuzumab.
The results disclosed herein indicate that ω-6 mono-unsaturated fatty acids, exemplified by trans-18:1n-9 (i.e., oleic acid or OA), ω-6 polyunsaturated fatty acids, exemplified by trans 18:3 n-6 (i.e., γ-linolenic acid or GLA), specifically suppress Her-2/neu overexpression which, in turn, interacts synergistically with anti-Her-2/neu breast cancer immunotherapy by promoting apoptotic cell death of breast cancer cells with an amplification of the Her-2/neu oncogene. See Table 1 for identification and characterization of fatty acids.
Her-2/neu (erbB-2) is one the most commonly analyzed oncogenes in breast cancer studies. This tyrosine kinase receptor regulates biological functions as diverse as cellular proliferation, transformation, differentiation, motility and apoptosis [52]. Therefore, modulation of Her-2/neu expression must be tightly regulated for normal cellular function. Consistent with this view, in vitro and animal studies demonstrate that deregulated Her-2/neu overexpression plays a pivotal role in oncogenic transformation, tumorigenesis and metastasis.
Her-2/neu overexpression occurs in about 20% of breast carcinomas and is associated with unfavorable clinical outcome and resistance to chemotherapy [53-56]. Little is known about the ultimate biochemical pathways through which fatty acids such as OA influence breast cancer risk and/or breast cancer progression. The results disclosed herein demonstrate that fatty acids (e.g., OA) can suppress Her-2/neu oncogene overexpression, representing a novel pathway through which individual dietary fatty acids modulate both the etiology and the aggressive behavior of cancer, such as breast cancer.
No toxicities have been reported or suspected with fatty acids such as OA consistent with use of one or more fatty acids as a dietary supplement, thereby providing a promising dietary intervention for the prevention and/or management of Her-2/neu-overexpressing carcinomas. Moreover, the data disclosed herein indicate further that dietary interventions based on, e.g., OA may be even more beneficial when given in combination with other therapies directed against Her-2/neu. Thus, OA co-exposure induces a dramatic increase in the sensitivity of Her-2/neu-overexpressing breast cancer cells to trastuzumab-induced cell growth inhibition upon anchorage-dependent and -independent conditions, and the nature of the interaction between OA and trastuzumab was found to be synergistic at clinically relevant trastuzumab concentrations. Importantly, exogenous supplementation with OA synergistically enhanced the ability of trastuzumab to induce down-regulation of p185Her-2/neu. Even without amplification of the Her-2/neu gene, moreover, the concurrent exposure to OA and trastuzumab was synergistically cytotoxic towards Her-2/neu-overexpressing cells by promoting DNA fragmentation associated with apoptotic cell death, as confirmed by TUNEL staining and cleavage of the caspase-3 substrate, PARP. The sensitizing effects of OA on trastuzumab efficacy were also accompanied by the up-regulation and nuclear accumulation of p27Kip1, a cyclin-dependent kinase inhibitor that plays a key role in the onset and progression of Her-2/neu-induced breast tumorigenesis that has recently been implicated in the development of trastuzumab resistance in breast cancer cells [42-44, 49-51]). Additionally, exogenous supplementation with OA significantly enhanced the ability of trastuzumab to inhibit the signaling pathways downstream of Her-2/neu that regulate cell cycle progression and/or cell death (i.e., AKT and MAPK).
As described in the following examples, flow cytometry, Western blotting, immunofluorescence microscopy, metabolic status (MTT), soft-agar colony formation, enzymatic in situ labeling of apoptosis-induced DNA double-strand breaks (TUNEL assay analyses), and caspase-3-dependent poly-ADP ribose polymerase (PARP) cleavage assays were used to characterize the effects of exogenous supplementation with a fatty acid of OA on the expression of the Her-2/neu oncogene, which plays an active role in breast cancer etiology and progression. In addition, the effects of administering a fatty acid (e.g., OA) on the efficacy of trastuzumab (Herceptin®), a humanized monoclonal antibody binding with high affinity to the ectodomain of the Her-2/neu-encoded p185Her-2/neu oncoprotein, were investigated. To study these issues, BT-474 and SKBr-3 breast cancer cells, which naturally exhibit amplification of the Her-2/neu oncogene, were used.
Flow cytometric analyses demonstrated a dramatic (up to 46%) reduction of cell surface-associated p185Her-2/neu following treatment of the Her-2/neu-overexpressing BT-474 and SK-Br3 cell lines with OA. This effect was comparable to that found following exposure to optimal concentrations of trastuzumab (up to 48% reduction with 20 mg/ml trastuzumab). Importantly, OA-induced suppression of Her-2/neu overexpression was not significantly prevented by the effective scavenger of reactive oxygen species vitamin E, thus ruling out that lipid peroxidation may be involved in this effect. Remarkably, the concurrent exposure to OA and suboptimal concentrations of trastuzumab (5 mg/ml) synergistically down-regulated Her-2/neu expression, as determined by flow cytometry (up to 70% reduction), immunoblotting, and immunofluorescence microscopy studies. The nature of the cytotoxic interaction between OA and trastuzumab revealed a strong synergism, as assessed by MTT-based cell viability and anchorage-independent soft-agar colony formation assays. Moreover, OA co-exposure synergistically enhanced trastuzumab efficacy towards Her-2/neu overexpressing cells by promoting DNA fragmentation associated with apoptotic cell death, as confirmed by TUNEL and caspase-3-dependent PARP cleavage. In addition, treatment with OA and trastuzumab dramatically increased both the expression and the nuclear accumulation of p27Kip1, a cyclin-dependent kinase inhibitor playing a key role in the onset and progression of Her-2/neu-related breast cancer. OA co-exposure also significantly enhanced the ability of trastuzumab to inhibit signaling pathways downstream of Her-2/neu, including phosphoproteins such as AKT and MAPK. Results indicate that OA is transcriptionally repressing Her-2/neu expression by up-regulating PEA3, an ets DNA-binding protein that inhibits Her-2/neu-promoted tumorigenesis by down-regulating Her-2/neu promoter activity [58-60]. These findings demonstrate that OA, the main mono-unsaturated fatty acid of olive oil, suppresses Her-2/neu overexpression which, in turn, interacts synergistically with anti-Her-2/neu immunotherapy by promoting the apoptotic cell death of breast cancer cells with Her-2/neu oncogene amplification. This previously unrecognized property of non-toxic fatty acids such as OA provides a molecular mechanism by which individual fatty acids regulate the malignant behavior of breast cancer cells, thereby providing methods for reducing the risk of developing any of a variety of erbB-2-overexpressing breast cancers, methods of treating such cancers, methods of maintaining the treatment of such cancers, and methods of mitigating or alleviating at least one symptom associated with such a cancer.
Cell Lines and Culture Conditions
The human breast cancer cell lines SK-Br3 and BT-474 were obtained from the American Type Culture Collection (ATCC), and they were routinely grown in phenol red-containing improved modified essential medium (IMEM; Biosource International, Camarillo, Calif.) containing 5% (v/v) heat-inactivated fetal bovine serum (FBS) and 2 mM L-glutamine. Cells were maintained at 37° C. in a humidified atmosphere of 95% air/5% CO2. Cells were screened periodically for Mycoplasma contamination.
Oleic acid (18:1n-9) and vitamin E (dl-α-tocopherol) were purchased from Sigma Chemical Co. (St Louis, Mo.). The cultures were supplemented, where indicated, with fatty acid-free bovine serum albumin (FA-free BSA; 0.1 mg/ml) complexed with a specific concentration of OA. A BSA-OA concentrate was formed by mixing 1 ml BSA (10 mg/ml) with various volumes (1-10 ml) of OA (200 mg/ml) in ethanol. The concentrate was mixed for 30 minutes at room temperature before addition to the cultures. Control cultures contained uncomplexed BSA. Trastuzumab (Herceptin®) is commercially available from Genentech, Inc.
The mouse monoclonal antibodies for p185Her-2/neu (Ab-3 and Ab-5 clones) were from Oncogene Research Products (San Diego, Calif.). Anti-β-actin goat polyclonal and anti-p27Kip1 rabbit polyclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz, Calif.). The anti-PARP p85 fragment antibody was from Promega Corp. (Madison, Wis.). Anti-MAPK, anti-phospho-MAPK, anti-AKT and anti-phospho-AKTSer473 rabbit polyclonal antibodies were from Cell Signal Technology (Beverly, Md.).
Flow Cytometry
Cells were seeded on 100-mm plates and cultured in complete growth medium. Upon reaching 75% confluence, the cells were washed twice with pre-warmed PBS and cultured in serum-free medium overnight. OA, trastuzumab, or a combination of OA plus trastuzumab as specified was added to the culture as specified, and incubation was carried out at 37° C. up to 48 hours in low-serum (0.1% FBS) medium. After treatment, cells were washed once with cold PBS and harvested in cold PBS. The cells were pelleted and resuspended in cold PBS containing 1% FBS. The cells were then incubated with an anti-p185Her-2/neu mouse monoclonal antibody (clone Ab-5) at 5 μg/ml for 1 hour at 4° C. The cells were then washed twice with cold PBS, resuspended in cold PBS containing 1% FBS, and incubated with a fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG secondary antibody (Jackson Immunoresearch Laboratories, West Grove, Pa.) diluted 1:200 in cold PBS containing 1% FBS for 45 minutes at 48° C. Finally, the cells were washed once in cold PBS, and flow cytometric analysis was performed using a FACScalibur flow cytometer (Becton Dickinson, San Diego, Calif.) equipped with Cell Quest Software (Becton Dickinson). The mean fluorescence signal associated with cells for labeled p185Her-2/neu was quantified using the GEO MEAN fluorescence parameter provided with the software.
Immunoblotting
Following treatments with OA, trastuzumab, or a combination of OA plus trastuzumab, as specified, cells were washed twice with PBS and then lysed in buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM B-glycer-olphosphate, 1 mM Na3VO4, 1 mg/ml leupeptin, 1 mM phenylmethylsulfonylfluoride] for 30 minutes on ice. The lysates were cleared by centrifugation in an Eppendorf tube (15 minutes at 14,000 r.p.m. at 4° C.). Protein content was determined against a standardized control using the Pierce protein assay kit (Rockford, Ill.). Equal amounts of protein were heated in SDS sample buffer (Laemli buffer) for 10 minutes at 70° C., subjected to electrophoresis on either 3-8% NuPAGE or 10% SDS-PAGE (p27Kip1, PARP, MAPK and AKT), and then transferred to nitrocellulose membranes. Non-specific binding on the nitrocellulose filter paper was minimized by blocking for 1 hour at room temperature (RT) with TBS-T [25 mM Tris-HCl, 150 mM NaCl (pH 7.5) and 0.05% Tween 20] containing 5% (w/v) non-fat dry milk. The treated filters were washed in TBS-T and then incubated overnight at 4° C. with specific primary antibodies in TBS-T/5% (w/v) BSA. The membranes were washed in TBS-T, horseradish peroxidase-conjugated secondary antibodies (Jackson Immunoresearch Laboratories) in TBS-T were added for 1 hour, and immunoreactive bands were detected by enhanced chemiluminescence reagent (Pierce). Blots were re-probed with an antibody for B-actin to control for protein loading and transfer. Densitometric values of protein bands were quantified using Scion Imaging Software (Scion Corp., Frederick, Md.).
In Situ Immunofluorescent Staining
Cells were seeded at a density of 1×104 cells/well in a four-well chamber slide (Nalge Nunc International, Rochester, N.Y.). Following treatments with OA, trastuzumab, or a combination of OA plus trastuzumab, as specified, cells were washed with PBS, fixed with 4% paraformaldehyde in PBS for 10 minutes, permeabilized with 0.2% Triton X-100/PBS for 15 minutes, and stored overnight at 4° C. with 10% horse serum in PBS. The cells were washed and then incubated for 2 hours with anti-p185Her-2/neu or anti-p27Kip1 antibodies, each separately diluted 1:200 in 0.05% Triton X-100/PBS. After extensive washes, the cells were incubated for 45 minutes with FITC-conjugated anti-mouse IgG (p185Her-2/neu) or tetramethylrhodamine isothiocyanate (TRITC)-conjugated anti-rabbit IgG (p27Kip1), each again separately diluted 1:200 in 0.05% Triton X-100/PBS. The cells were washed five times with PBS and mounted with VECTASHIELD+DAPI (Vector Laboratories, Burlingame, Calif.). As controls, cells were stained with primary or secondary antibody alone. Control experiments did not display significant fluorescence in any case. Indirect immunofluorescence was recorded on a Zeiss microscope. Images were noise-filtered, corrected for background and prepared using Adobe Photoshop.
Anchorage-dependent Cell Proliferation
SK-Br3 and BT-474 cells exponentially growing in IMEM-5% FBS were trypsinized and re-plated in 24-well plates at a density of 10,000 cells/well. Cells were incubated for 24 hours to allow for attachment, after which a zero time point was determined. Cells were treated with OA, trastuzumab, or a combination of OA plus trastuzumab, as specified. Cell number was determined at days 0, 3 and 6 with a Coulter Counter (Coulter Electronics, Inc., Hialeah, Fla.). All assays were performed at least three times in triplicate. The data are presented as mean of number cells 104/well±SD.
In Vitro Chemosensitivity Testing
Trastuzumab sensitivity was determined using a standard colorimetric MTT (3-4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide) reduction assay. Cells in exponential growth were harvested by trypsinization and seeded at a concentration of 5×103 cells/200 μl/well into 96-well plates, and incubated overnight for attachment. The medium was then removed and fresh medium, along with various concentrations of trastuzumab, OA or combinations of compounds, was added to cultures in parallel. Agents were studied in combination concurrently. Control cells without agents were cultured using the same conditions with comparable media changes. Compounds were not renewed during the entire period of cell exposure. Following treatment, the medium was removed and replaced with fresh drug-free medium (100 μl/well), and MTT (5 mg/ml in PBS) was added to each well at a volume of 1:10. After incubation for 2-3 hours at 37° C., the supernatants were carefully aspirated, 100 ml of DMSO were added to each well, and the plates were agitated to dissolve the crystal product. Absorbances were measured at 570 nm using a multi-well plate reader (Model Anthos Labtec 2010 1.7 reader). The cell viability effects from exposure of cells to each compound alone and to their combination were analyzed, generating concentration-effect curves as a plot of the fraction of unaffected (surviving) cells versus drug concentration. Dose-response curves were plotted as percentages of the control cell absorbances, which were obtained from control wells treated with appropriate concentrations of the compound vehicles that were processed simultaneously. For each treatment, cell viability was evaluated as a percentage using the following equation: (A570 of treated sample/A570 of untreated sample)×100. Drug sensitivity was expressed in terms of the concentration of drug required for a 50% reduction of cell viability (IC50). Since the percentage of control absorbance was considered to be the surviving fraction of cells, the IC50 values were defined as the concentration of drug that produced a 50% reduction in control absorbance (by interpolation). The degree of sensitization to trastuzumab by OA was evaluated by dividing IC50 values of control cells by those obtained when cells were exposed to OA during exposure to trastuzumab.
Determination of Synergism: Isobologram Analysis
The interaction between OA and trastuzumab was evaluated using the isobologram technique [45], a dose-oriented geometric method of assessing drug interactions. With the isobologram method, the concentration of one agent producing a desired (e.g. 50% inhibitory) effect is plotted on the horizontal axis and the concentration of another agent producing the same degree of effect is plotted on the vertical axis. A straight line joining these two points represents zero interaction (addition) between two agents. The experimental isoeffect points are the concentrations (expressed relative to the IC50 concentrations) of the two agents that, when combined, kill 50% of the cells. When the experimental isoeffect points fall below that line, the combination effect of the two drugs is considered to be supra-additive or synergistic, whereas antagonism occurs if the point lies above the line. A quantitative index of these interactions was provided by the isobologram equation: CIx=(a/A)+(b/B), where, for this study, A and B represent the respective concentrations of OA and trastuzumab required to produce a fixed level of inhibition (IC50) when administered alone, a and b represent the concentrations required for the same effect when the drugs were administered in combination, and CIx represents an index of drug interaction (interaction index). Ix values<1 indicate synergy, a value of 1 represents addition, and values of>1 indicate antagonism.
Soft-agar Colony-formation Assays
The efficiency of colony formation in liquid culture was determined by monitoring anchorage-independent cell growth in soft-agar experiments. A bottom layer of 1 ml IMEM containing 0.6% agar and 10% FBS was prepared in 35-mm multi-well cluster dishes. After the bottom layer solidified, cells (10,000/dish) were added in a 1 ml top layer containing OA, trastuzumab, a combination of OA plus trastuzumab, or vehicles (v/v) in 0.35% agar and 10% FBS, as specified. All samples were prepared in triplicate. Dishes were incubated in a humidified 5% CO2 incubator at 37° C., and colonies measuring >50 mm were counted 14 days after staining with nitroblue tetrazolium (Sigma) using a cell colony counter (Ommias 3600; Imaging Products International, Inc., Charley, Va.).
Apoptosis
Detection of apoptosis in SK-Br3 and BT-474 cells treated with OA, trastuzumab, or a combination of OA plus trastuzumab, as specified, was performed by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling (TUNEL) analysis using the DeadEnd® Fluorometric TUNEL System (Promega Inc.) according to the manufacturer's instructions. Briefly, cells were split at a density of 2×104 cells/well in an eight-well chamber slide (Lab-Tek). After 48 hours incubation the cells were treated with trastuzumab in the absence or presence of OA for 72 hours. Following treatment, cells were washed twice with PBS and fixed with 4% methanol-free paraformaldehyde for 10 minutes. Cells were washed twice more with PBS and permeabilized with 0.2% Triton X-100 for 5 minutes. After two more washes, each slide was covered with equilibration buffer for 10 minutes. The buffer was then aspirated, and the slides were incubated with TdT buffer at 37° C. for 1 hour. The reaction was stopped with 2×standard saline citrate and the slides were viewed under an immunofluorescence microscope (Zeiss). Apoptosis was quantified by determining the proportion of cells containing nuclei with complete TUNEL-associated staining. One hundred cells were assessed in triplicate for each treatment.
Statistical Analysis
Statistical analysis of mean values was performed using the nonparametric Mann-Whitney test. Differences were considered significant at P<0.05 and P<0.005.
To assess the effects of exogenous supplementation with a fatty acid (OA) on Her-2/neu expression, SK-Br3 and BT-474 cells, after a 24-hour starvation period in medium without serum, were incubated for 48 hours with 10 mM of OA complexed to BSA in low-serum (0.1% FBS) conditions. The cell surface-associated expression of Her-2/neu-encoded p185Her-2/neu oncoprotein was then determined by measuring the binding of a mouse monoclonal antibody directed against the ectodomain of p185Her-2/neu (Ab-5 clone) in OA-treated BT-474 and SK-Br3 cells. Flow cytometric analysis of cell surface-associated p185Her-2/neu demonstrated a significant reduction of p185Her-2/neu expression levels in BT-474 breast cancer cells following OA treatment (up to 46% reduction at 10 mM OA;
The down-regulatory effects of the fatty acid OA on p185Her-2/neu expression indicate that exogenous supplementation with OA may sensitize breast cancer cells to the well-known p185Her-2/neu down-regulatory actions of trastuzumab [46-48]. To assess this effect, cell surface-associated p185Her-2/neu was first measured by flow cytometry following treatment with low doses of OA (5 mM) and trastuzumab (5 mg/ml). Remarkably, the concurrent combination of OA and trastuzumab reduced p185Her-2/neu expression more than when either agent was administered alone (
The impact of OA supplementation in the subcellular localization of p185Her-2/neu in Her-2/neu-overexpressing breast cancer cells was also investigated. To address this question, BT-474 cells, at 48 hours after treatment with OA, trastuzumab, or OA plus trastuzumab, were permeabilized with Triton X-100 for the intracellular delivery of antibodies. Thereafter, p185Her-2/neu cellular localization was assessed using the anti-erbB2, Ab-3 mouse monoclonal antibody (Oncogene Research Products), which is directed against the C-terminal 14 amino acids of p185Her-2/neu. Untreated BT-474 cells showed a prominent cell-surface staining of p185Her-2/neu whereas, upon OA treatment, p185Her-2/neu-associated membrane staining was markedly reduced (
The data described in this Example and in other Examples is entered on the effects of oleic acid, an exemplary fatty acid of the ω-9 monounsaturated class. Other fatty acids conforming to the general definition of useful fatty acids are also known and have been shown to be effective. See Table 2.
The effects of a concurrent combination of OA and trastuzumab on the anchorage-dependent growth properties of Her-2/neu-overexpressing breast cancer cells were also assessed. As expected, the anchorage-dependent cell growth of Her-2/neu overexpressing was significantly decreased in the presence of increasing concentrations of trastuzumab, while exogenous supplementation with low concentrations of OA had no notable effects on breast cancer cell proliferation. Interestingly, when added in the presence of OA, trastuzumab further inhibited cell proliferation of SK-Br3 and BT-474 cells (FIG. 3A, left panels). Moreover, the increase in growth inhibition with the addition of OA over that of trastuzumab itself was statistically significant (
Next, the cytotoxic interactions between OA and trastuzumab and their effects on SK-Br3 and BT-474 cells were examined by evaluating the metabolic status of breast cancer cells co-treated with trastuzumab and OA, as judged by the mitochondrial conversion of the tetrazolium salt, MTT, to its formazan product (MTT assay). First, we measured the changes in cell toxicity of 10 μg/ml trastuzumab after 72 hours of co-exposure to increasing concentrations of OA. The simultaneous presence of OA during the incubation period with trastuzumab caused a significant increase in the cytotoxic effects of trastuzumab (
The acquisition of anchorage-independent growth is generally considered to be one of the in vitro properties associated with the malignancy of cells. In fact, colonization of metastatic tumor cells at a distant site may be partially modeled in soft-agar assays. Therefore, the effects of concurrent exposure to trastuzumab and OA on the ability of Her-2/neu overexpressing cells to grow in anchorage-independent conditions were evaluated. As a single agent, OA slightly decreased the ability of SK-Br3 and BT-474 cells to form colonies in soft agar, whereas trastuzumab, as expected, significantly blocked anchorage-independent growth of Her-2/neu overexpressing cells (
To assess if the synergistic interaction between trastuzumab and OA observed above represented cell death, we next focused on an apoptotic effect of the combination of OA and trastuzumab as measured by the enzymatic in situ labeling of apoptosis-induced DNA double-strand breaks (TUNEL assay). Individually, OA (5 μM) and trastuzumab (10 μg/ml) caused slight increases in the number of apoptotic cells (2% and 16% of TUNEL-positive cells, respectively). Remarkably, there was an impressive increase in apoptosis when BT-474 cells were treated simultaneously with both agents (51% TUNEL-positive cells;
The signaling pathways downstream of Her-2/neu that regulate cell cycle progression and/or cell death were investigated to assess whether they were modified by OA. The treatment of cancer cells with trastuzumab results not only in down-regulation of p185Her-2/neu, but also in further downstream cellular events, including accumulation of the cyclin-dependent kinase inhibitor p27Kip1 [42-44, 49]. The cyclin-dependent kinase inhibitor (CDKi) p27Kip1 plays a key role in the onset and progression of Her-2/neu-induced breast tumorigenesis and breast cancer progression, and is further involved in the development of trastuzumab resistance [42, 43, 49-51]. A slight increase in the expression of p27Kip1 was observed after the treatment of BT-474 cells with suboptimal concentrations of OA. The expression of p27Kip1 was significantly enhanced in the presence of trastuzumab. Remarkably, a dramatic up-regulation of p27Kip1 expression was observed in trastuzumab-treated BT-474 cells in the presence of increasing concentrations of OA (
The effect of an OA-induced interruption of Her-2/neu-dependent signaling on the cellular localization of p27Kip1 was also evaluated. Using immunofluorescence microscopy, most of the p27Kip1 was found in the cytosol of proliferating BT-474 cells. Treatment with suboptimal concentrations of trastuzumab resulted in a significant translocation of immunufluorescent p27Kip1 from cytosol to cell nuclei, while co-treatment with OA resulted in an almost complete translocation of p27Kip1 from cytosol to cell nuclei (
The signaling pathways down-stream of Her-2/neu that regulate cell cycle progression and/or cell death were examined for effects following exogenous supplementation with OA. In BT-474 cells, OA treatment inhibited active MAPK and active AKT as measured by antibodies specific to phospho-MAPK and phospho-Ser473 AKT, respectively, without changes in total MAPK and total AKT (
The ω-6 poly-unsaturated fatty acid γ-linolenic acid (GLA; 18:3n-6) was analyzed for a possible effect on the expression of the Her-2/neu (erbB-2) oncogene, which is involved in development of numerous types of human cancer. Flow cytometric and immunoblotting analyses demonstrated that GLA treatment substantially reduced Her-2/neu protein levels in the Her-2/ neu-overexpressing cell lines BT-474, SK-Br3, and MDA-MB-453 (breast cancer), SK-OV3 (ovarian cancer), and NCI-N87 (gastrointestinal tumor derived). GLA exposure led to a dramatic decrease in Her-2/neu promoter activity and a concomitant increase in the levels of polyomavirus enhancer activator 3 (PEA3), a transcriptional repressor of Her-2/neu, in these cell lines. In transient transfection experiments, a Her-2/neu promoter bearing a mutated PEA3 site was not subject to negative regulation by GLA in Her-2/neu-overexpressing cell lines. Concurrent treatments of Her-2/neu-overexpressing cancer cells with GLA and the anti-Her-2/neu antibody trastuzumab led to synergistic increases in apoptosis as well as reduced growth and colony formation.
The oil from seeds of the evening primrose (and that from seeds of borage and black currant) contains γ-linolenic acid (GLA), a member of the ω-6 family of polyunsaturated fatty acids.
Exogenous supplementation of cultured breast cancer cells with GLA significantly diminished proteolytic cleavage of the extracellular domain of the Her-2/neu-coded p185Her-2/neu tyrosine kinase oncoprotein and, consequently, its activation (16) . Considering that activation and overexpression of the Her-2/neu oncogene are crucial for the etiology, progression, and cell sensitivity to various anti-cancer treatments in about 30% of breast carcinomas (17-32), these findings showed a previously unrecognized mechanism by which GLA might regulate breast cancer cell growth, metastasis formation, and response to chemotherapy and endocrine therapy. Two remaining issues are, first, whether GLA-induced deactivation of p185Her-2/neu relates to GLA-induced changes in Her-2/neu gene expression; and, second, whether the ability of GLA to regulate the Her-2/neu oncogene is a common mechanism of GLA's action against other types of cancer.
To characterize the effects of GLA on the expression of the Her-2/neu oncogene, BT-474 and SK-Br3 breast cancer cells, which naturally contain amplified copies of the Her-2/neu oncogene (33, 34), were first treated with GLA (10 μg/mL for 48 hours). In flow cytometry analyses, levels of cell surface-associated Her-2/neu protein, i.e., p185Her-2/neu were substantially lower in GLA-treated cells than in vehicle-treated cells (
Reporter gene expression and reverse transcription-polymerase chain reaction (RT-PCR) analyses were undertaken to characterize the effects of GLA on the transcription of the Her-2/neu gene. Treatment of BT-474 and SK-Br3 cells that had been transfected with a construct containing a luciferase reporter gene driven by a wild-type Her-2/neu promoter fragment with GLA (10 μg/mL for 48 hours) led to a strong reduction in reporter gene expression in both lines (Table 3).
For semiquantitative RT-PCR analyses, BT-474 and SK-Br3 cells were treated with varying concentrations of GLA (5, 10, or 20 μg/ml for 48 hours) and then total RNA was extracted from the cells. One microgram of total RNA was then reverse-transcribed and amplified with specific primers for Her-2/neu, and the products were separated on agarose gels (
Medication of the GLA-induced repression of Her-2/neu transcription is mediated by the DNA binding protein PEA3 was also investigated. PEA-3, a member of the Ets transcription factor family, specifically targets a DNA sequence in the Her-2/neu promoter (and not in the promoter for genes encoding any other HER isoform), thus suppressing Her-2/neu overexpression in cancer cells (36-38). Immunoblot analysis of BT-474 and SK-Br3 cells that were treated for 48 hours with 10 μg/mL GLA showed an increase in the levels of PEA3 protein in both cell lines relative to levels in control-treated cells (
The luciferase activities reported in Table 3 were assayed in cells that were transiently transfected with a pGL2-luciferase (Promega Inc., Madison, Wis.) construct containing a luciferase reporter gene under the control of a Her-2/neu promoter fragment containing a wild-type or mutant PEA3 binding site, as previously described by Xing et al. (36), incorporated herein by reference. Cells were transfected using FuGENE 6 transfection reagent (Roche Biochemicals, Indianapolis, Ind.) as directed by the manufacturer and were treated with vehicle (ethanol; EtOH) or γ-linolenic acid (10 μg/mL for 24 hours). Luciferase activity from cell extracts was detected with a Luciferase Assay System (Promega Inc.). Results for all treatments are given as the percentage of luciferase activity relative to that in vehicle-treated cells transfected with the wild-type promoter construct. Data are the means and 95% confidence intervals (CIs) of five experiments, each performed in triplicate.
To examine whether the increased PEA3 levels might mediate the inhibition of Her-2/neu transcription in GLA-treated cells, the effect of GLA on transcription from a Her-2/neu promoter bearing a mutated PEA3 binding sequence, at −33 to −28, that is known to abolish PEA3 binding (36, incorporated herein by reference) was investigated. For this analysis we used the same luciferase reporter gene construct described above but containing the HER-2/neu promoter mutation at the PEA3 binding site (5′-GAG GAA-3′ at −33 to −28 changed to 5′-GAG CTC-3′). The mutant promoter was much less active than the wild-type promoter in untreated control cells (Table 3), consistent with a PEA3 binding site on the Her-2/neu promoter acting as a positive regulatory element necessary for elevated expression of the Her-2/neu oncogene in cancer cells. In addition, transcription from the mutant promoter was not reduced in GLA-treated cells.
To gain insight into the possible role of PEA3 in GLA-mediated repression of Her-2/neu transcription, Her-2/neu and PEA3 protein levels were evaluated by immunoblot analysis in a panel of cancer cells with high or low levels of endogenous Her-2/neu (
The influence of GLA-induced transcriptional repression of Her-2/neu on the growth-inhibitory effects of trastuzumab, a humanized monoclonal antibody that binds with high affinity to Her-2/neu and has therapeutic effects in patients with Her-2/neu-positive breast cancer (39-41), was also investigated. For these analyses, apoptosis in BT-474 cells treated with GLA and/or trastuzumab was determined by terminal deoxynucleotidyltransferase-mediated dUTPbiotin nick end labeling (TUNEL) using the DeadEnd Fluorometric TUNEL System (Promega Inc., Madison, Wis.). Immunofluorescence microscopy revealed many strongly positive nuclei in BT-474 cells treated with both drugs, whereas such nuclei were rare in untreated, GLA-treated, and trastuzumab-treated cells (
The synergism was also revealed in 3,4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT)—based cell viability assays and by isobologram analysis. Concurrent administration of 10 μg/ml GLA increased the sensitivity of BT-474 and SK-Br3 cells to trastuzumab by approximately 30- and 40-fold, respectively, and two-way ANOVAs showed that these increases were statistically significant. This combination yielded combination index 50 (CI50) values of 0.697 and 0.615 in BT-474 and SK-Br3 cells, respectively, thus suggesting that the interaction was truly synergistic (CI50=1, additive; CI50<1, synergism). When soft-agar assays were used to investigate the actions of GLA on Her-2/neu- induced anchorage-independent cancer cell growth (23, 24), a two-way ANOVA showed that GLA cotreatment enhanced the growth-inhibitory effects of suboptimal doses of trastuzumab in a statistically significant manner in both BT-474 and SK-Br3 breast cancer cell lines (see
Together, the results indicate that GLA-promoted accumulation of PEA3, a potent repressor of the Her-2/neu promoter (36-38), is a key mechanism underlying GLA-induced suppression of Her-2/neu overexpression in cancer cells. It is possible that GLA activation of other factors that interact with the Her-2/neu promoter, such as AP-2, may account for the reduced Her-2/neu promoter activity in GLA-treated cells; however, AP-2-regulated Her-2/neu promoter regions have different roles in breast and non-breast cancer cells (42-45) and we found that GLA does not appear to regulate AP-2 expression. Considering that GLA mitigates Her-2/neu overexpression by affecting PEA3 binding to the Her-2/ neu promoter, the anti-Her-2/neu actions of GLA should not be affected by the mechanisms of resistance described for trastuzumab (46, 47). Therefore, the disclosed results establish that GLA-induced transcriptional repression of the Her-2/neu oncogene provides a molecular approach to treating Her-2/ neu-overexpressing carcinomas, e.g., in combination with trastuzumab.
Transient transfection experiments with the human Her-2/neu promoter-driven luciferase gene revealed that OA represses Her-2/neu gene expression in tumor-derived cells exhibiting Her-2/neu gene amplification and overexpression, including SK-Br3 (≦56% reduction), SK-OV3 (≦75% reduction) and NCI-N87 (55% reduction) breast, ovarian and stomach cancer cell lines, respectively. Also, marginal decreases in promoter activity were observed in cancer cells expressing physiological levels of Her-2/neu (<20% reduction in MCF-7 breast cancer cells). Remarkably, OA treatment in Her-2/neu-overexpressing cancer cells was found to induce up-regulation of the Ets protein Polyomavirus Enhancer Activator 3 (PEA3), a transcriptional repressor of Her-2/neu that binds to a PEA3 binding site in the Her2/neu promoter. Also, an intact PEA3 DNA binding site in the endogenous Her-2/neu gene promoter was essential for OA-induced repression of the endogenous gene. Moreover, OA treatment failed to decrease Her-2/neu protein levels in MCF-7/Her2-18 transfectants, which stably express full-length human Her-2/neu cDNA controlled by a SV40 viral promoter. Thus, unsaturated fatty acids such as OA are expected to lower the risk of malignant neoplasms, especially breast and stomach cancer, but also in ovary, colon and endometrium cancer {1-10}.
The preceding examples establish that exogenous supplementation of cultured breast cancer cells with OA significantly down-regulated the expression of Her-2/neu {13}. These findings are significant, in part because no toxicities have been reported or suspected with OA, and supplementation with OA therefore represents a promising dietary intervention for the prevention and/or management of Her-2/neu-related breast, and other, carcinomas {27, 28}. The results provided in this Example show that an unsaturated fatty acid such as OA downregulates Her-2/neu by a mechanism relevant to types of cancer other than breast cancer.
Although overexpression of Her-2/neu both in tumors and in derived cell lines was originally attributed solely to amplification of the erbB-2 gene (usually 2- to 10-fold), an elevation in Her-2/neu mRNA levels per gene copy is also observed in all the cell lines examined exhibiting gene amplification {29}. This indicates that overexpression of the gene precedes and increases the likelihood of gene amplification. Indeed, an increase in transcription rate sufficient to account for the degree of overexpression has been shown in a number of Her-2/neu-overexpressing cancer cell lines {30}. The experiments described in this Example sought to characterize the effects of OA treatment on the transcription rate of Her-2/neu gene. Also addressed is whether the ability of OA to down-regulate Her-2/neu occurs by a common mechanism of OA action towards tumor types reported to exhibit Her-2/neu overexpression, including breast, ovarian and gastric carcinomas. The data show that OA promoted the up-regulation of PEA3, the potent trans-repressor of the human Her-2/neu gene. This up-regulation accounts, at least in part, for the ability of OA to suppress Her-2/neu overexpression in cancer cells. OA-induced transcriptional repression of the Her-2/neu gene is operative in various types of human malignancies, such as breast, ovarian, and stomach carcinomas.
Phenol red-containing Improved Minimal Essential Medium (IMEM) was from Biofluids (Rockville, Md., USA). Oleic acid (18:1n-9) was purchased from Sigma Chemical Co. (St. Louis, Mo., USA). The cultures were supplemented, where indicated, with fatty acid-free bovine serum albumin (FA-free BSA; 0.1 mg/ml) complexed with a specific concentration of OA. A BSA-OA concentrated (100×) solution was formed by mixing 1 ml BSA (10 mg/ml) with various volumes (1-10 μl) of OA (200 mg/ml) in ethanol. The concentrate was mixed for 30 minutes at room temperature before addition to cultures. Control cultures contained uncomplexed BSA.
The primary antibody for Her-2/neu immunoblotting was an anti-p185Her-2/neu mouse monoclonal antibody from Oncogene Research Products (Clone Ab-3; San Diego, Calif., USA). Anti-PEA3 mouse monoclonal (sc-113) and anti-β-actin goat polyclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz, Calif., USA).
SK-Br3 (breast cancer), MDA-MB-231 (breast cancer), SK-OV3 (ovarian cancer), and NCI-N87 (gastrointestinal cancer) cell lines were obtained from the American Type Culture Collection (ATCC). MCF-7 cells stably overexpressing the Her-2/neu oncogene (MCF-7/Her2-18) were generated using conventional techniques. Cells were routinely grown in IMEM containing 5% (v/v) heat-inactivated fetal bovine serum (FBS) and 2 mM L-Glutamine. Cells were maintained at 37° C. in a humidified atmosphere of 95% air/5% CO2. Cells were screened periodically for Mycoplasma contamination.
To conduct Her-2/neu promoter activity assays, cells were initially transfected using FuGENE 6 transfection reagent (Roche Biochemicals, Indianapolis, Ind.) as directed by the manufacturer. Overnight-serum starved cancer cells seeded into 24-well plates (˜5×104 cells/well) were transfected in low-serum (0.1% FBS) media with 1.5 μg/well of the pGL2-luciferase (Promega, Madison, Wis.) construct containing a luciferase reporter gene driven by either an intact (Her-2/neu wild-type PEA3-binding site-luciferase) or by a mutated (Her-2/neu mutated PEA3-binding site-luciferase) Her-2/neu promoter fragment along with 150 μg/well of the internal control plasmid pRL-CMV, which was used to correct for transfection efficiency. After 18 hours, the transfected cells were washed and incubated with either ethanol (v/v) or 20 μM OA in 0.1% FBS. Approximately 24 hours after treatments, luciferase activities from cell extracts were detected with a luciferase Assay System following manufacturer's instructions (Promega, Madison, Wis., USA) using a Victor2™ Multilabel Counter (Perkin-Elmer Life Sciences).
Following treatments with OA, cells were washed twice with PBS and then lysed in buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM α-glycerolphosphate, 1 mM Na3VO4, 1 μg/ml leupeptin, 1 mM phenylmethylsulfonylfluoride] for 30 minutes on ice. The lysates were cleared by centrifugation in an eppendorf tube (15 minutes at 14,000 rpm at 4° C.). Protein content was determined against a standardized control using the Pierce protein assay kit (Rockford, Ill.). Equal amounts of protein were heated in SDS sample buffer (Laemmli) for 10 minutes at 70° C., subjected to electrophoresis on either 3-8% NuPAGE Tris-Acetate (p185Her-2/neu) or 10% SDS-PAGE (PEA3) and then transferred to nitrocellulose membranes. Non-specific binding on the nitrocellulose filter paper was minimized by blocking for 1 hour at room temperature (RT) with TBS-T [25 mM Tris-HCl (pH 7.5), 150 mM NaCl and 0.05% Tween-20] containing 5% (w/v) non-fat dry milk. The treated filters were washed in TBS-T and then incubated with primary antibodies for 2 hours at room temperature in TBS-T containing 5% (w/v) non-fat dry milk. The membranes were washed in TBS-T, horseradish peroxidase-conjugated secondary antibodies in TBS-T were added for 45 minutes, and immunoreactive bands were detected by enhanced chemiluminescence reagent (Pierce, Rockford, Ill.). Blots were re-probed with an anti-β-actin goat polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.) to control for protein loading and transfer. Densitometric values of protein bands were quantified using Scion Imaging Software (Scion Corp., Frederick, Md.).
Data are the mean and 95% confidence intervals (95% CI) of three independent experiments. A two-way ANOVA was used to analyze differences in the percentage of luciferase activity between the treatment and the control groups.
Exogenous supplementation with OA inhibited Her-2/neu gene promoter activity in breast, ovarian and stomach cancer cells.
Reporter gene expression was used to characterize the effects of OA on Her-2/neu oncogene transcription. We performed transient transfection experiments with a luciferase reporter gene driven by the wild-type Her-2/neu promoter (pNulit;
Although the precise molecular mechanisms governing Her-2/neu promoter activity in Her-2/neu-overexpressing cancer cells are far from being totally defined, two main groups of transcription factors, namely the AP-2 and the Ets families of transcription factors, have been shown to be both required for maximal Her-2/neu promoter activity and to be associated with overexpression of the gene in cancer {31}. Recent studies showed that AP-2-regulated Her-2/neu promoter regions have different roles in breast and non-breast cancer cells {31-34}, while the Ets sites appear to be non-tissue specific (“universal”) regulators of Her-2/neu promoter activity {31, 35-36}. In the absence of exogenous supplementation with OA, Her-2/neu-overexpressing SK-Br3, SK-OV3 and NCI-N87 cancer cells did express undetectable to low levels of the DNA-binding protein PEA3, a member of the Ets transcription factor family. PEA3 specifically targets a DNA sequence in the Her-2/neu promoter that suppresses Her-2/neu overexpression and inhibits Her-2/neu-dependent tumorigenesis {35, 36}. Interestingly, a significant up-regulation of PEA3 protein expression, concomitantly with down-regulation of Her-2/neu protein expression, occurred following OA treatment in SK-Br3, SK-OV3 and NCI-N87 cancer cells (
OA-induced transcriptional repression of the Her-2/neu gene requires an intact PEA3 binding site at the endogenous Her-2/neu promoter. To examine whether the increased PEA3 levels might mediate the inhibition of Her-2/neu transcription in OA-treated Her-2/neu-overexpressing cancer cells, the effects of OA on transcription from a promoter bearing a mutated PEA3 binding site (at −33 to −28) that is known to abolish PEA3 binding {35, incorporated herein by reference} were examined. For this analysis the same luciferase reporter gene construct described above, but containing a Her-2/neu promoter mutation at the PEA3 binding site (5′-GAGGAA GAGGAA-3′ (SEQ ID NO: 3) to 5′-GAGCTC-GAGCTC-3′) (
The above findings indicated that the formation of inhibitory “PEA3 protein-PEA3 DNA binding site” complexes at the endogenous Her-2/neu promoter could be required for OA-induced transcriptional repression of Her-2/neu gene in Her-2/neu-overexpressing cancer cells. This realization was further supported when the effects of OA treatment on Her-2/neu protein expression, Her-2/neu promoter activity, and PEA3 accumulation were characterized in MCF-7 breast cancer cells, which naturally express physiological (i.e., unelevated) levels of Her-2/neu, and MCF-7 cells engineered to overexpress Her-2/neu under the transcriptional control of a different promoter (i.e., MCF-7/Her2-18 stable transfectants expressing full-length human Her-2/neu cDNA under SV40 promoter control). MCF-7/Her2-18 cells are known to express 45-fold more Her-2/neu than parental MCF-7 cells or the MCF-7/neo control sub-line expressing a neomycin phosphotransferase gene {37}. Her-2/neu and PEA3 protein levels in MCF-7/neo cells were not significantly affected by exogenous supplementation with OA, while the luciferase reporter activity of the wild-type Her-2/neu promoter was slightly reduced by either OA treatment (up to 12% reduction;
The data provided herein demonstrate that: i) the PEA3 binding motif in the Her-2/neu promoter functions as a positive regulatory element for Her-2/neu gene transcription solely in cancer cells naturally exhibiting both Her-2/neu gene amplification and Her-2/neu protein overexpression (
Overexpression of the Her-2/neu oncogene is a frequent molecular event in multiple human cancers. Her-2/neu codes for a transmembrane tyrosine kinase orphan receptor p185Her-2/neu that regulates biological functions as diverse as cellular proliferation, differentiation, motility and apoptosis. Therefore, modulation of Her-2/neu levels must be tightly regulated for normal cellular function. Consistently, in vitro and animal studies demonstrate that deregulated Her-2/neu expression plays a pivotal role in malignant transformation, tumorigenesis and metastasis. Patients with Her-2/neu-overexpressing cancer cells are associated with unfavorable prognosis, shorter relapse time, and low survival rate {14-26}.
The data disclosed herein establish that OA-promoted accumulation of PEA3, the potent trans-repressor of the human Her-2/neu promoter, is a key molecular feature that accounts, at least in part, for the down-regulatory effects of OA on the expression of the Her-2/neu oncogene in cancer cells. The data do not prove, however, that exogenous supplementation with OA exclusively suppresses Her-2/neu overexpression via PEA3. Other Her-2/neu promoter interacting factors, such as AP-2, a member of a family of highly homologous proteins all of which can activate the Her-2/neu promoter {31-34}, may also contribute to the blockade of Her-2/neu promoter activity observed upon OA exposure. While recent studies suggest that AP-2 is not a major player in the increased levels of Her-2/neu transcripts in colon and ovary cancer cells, thus suggesting that the promoter regions leading to Her-2/neu overexpression are different in breast and non-breast cancer cells {34}, no effects of OA on AP-2 levels in Her-2/neu-overexpressing cancer cells were observed. Considering that OA exposure similarly impaired Her-2/neu promoter activity and concomitantly up-regulated PEA3 expression in all the Her-2/neu-overexpressing cell models evaluated, these results support the view that PEA3 and its Ets binding site within the Her-2/neu promoter are the main down-stream effectors involved in OA-induced repression of Her-2/neu oncogene expression in cancer cells, including breast, ovarian and stomach cancer cells (
The data presented above established that the combined treatment with OA and trastuzumab (Herceptin™), a monoclonal antibody that targets the extracellular domain of Her-2/neu, synergistically increased the extent of apoptotic cell death in Her-2/neu overexpressing cells and strongly impaired the ability of Her-2/neu-overexpressing cancer cells to grow under anchorage-independent conditions. Considering that OA mitigates Her-2/neu overexpression by affecting PEA3 binding to the Her-2/neu promoter, this mechanism of action would not be affected by the mechanisms of resistance described for trastuzumab-based anti-Her-2/neu immunotherapy {46, 47}. The ability of OA to transcriptionally repress Her-2/neu overexpression in a PEA3-dependent manner operates equally in various types of human malignancies, including breast, ovarian and stomach/colon carcinomas.
Each of the following references, cited throughout this disclosure, is incorporated by reference herein in its entirety.
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The present application claims the benefit of priority of U.S. Provisional Application No. 60/757,926, which was filed Jan. 10, 2006 and is specifically incorporated herein by reference in its entirety.
This invention was made with U.S. government support and the U.S. government may have certain rights in the invention pursuant to the terms of Grant No. BC033538 from the Dept. of Defense and Grant No. P50CA89018-03 from the National Institutes of Health.
| Number | Date | Country | |
|---|---|---|---|
| 60757926 | Jan 2006 | US |
