Patent Application
20150167050
It has been discovered that the activity of a particular enzyme, polyisoprenylated methylated protein methyl esterase (PMPMEase which is also known as human carboxylesterase 1 or hCE1), is elevated in certain cancers, such as in breast cancer. The present invention is methods for the diagnosis and treatment of cancer, by measuring the activity of the enzyme PMPMEase.
Breast cancer is not one disease but several different diseases. Three types of breast cancer are estrogen receptor (ER)-positive breast cancer, progesterone receptor (PR)-positive breast cancer (together termed hormone receptor positive breast cancer), and human epidermal growth factor receptor 2 (HER2)-positive breast cancer (triple positive breast cancer refers to the presence of ER, PR, and HER2 receptors). These subtypes of breast cancer are generally diagnosed based upon the presence, or lack of these three receptors. In addition, the most successful treatments for breast cancer target these receptors. Another type of breast cancer is called triple negative breast cancer, since none of these three receptors are found in the offending tumor. Because of its triple negative status, triple negative tumors generally do not respond to receptor targeted treatments. Depending on the stage of its diagnosis, triple negative breast cancer can be particularly aggressive, and more likely to recur than other subtypes of breast cancer. Although it cannot be treated with receptor targeted treatments it is commonly receptive to chemotherapy. The lack of targets for treating triple negative breast cancer implies that more research needs to be pursued to find what drives this form of cancer as well as other cancers of unknown etiologies.
Proper diagnosis of the type of breast cancer is essential for proper treatment.
Protein polyisoprenylation and subsequent methylation are essential modifications on a significant proportion of eukaryotic proteins. The modifications are a series of post-translational modifications involving motifs such as -CAAX wherein C is cysteine, A is any aliphatic amino acid, and X is any amino acid whose nature specifies either farnesylation or geranylgeranylation. The modifications include polyisoprenylation of the cysteine of the -CAAX motif (on the sulfur), proteolysis of the carboxyl-terminal three amino acids (AAX), and methylation of the carboxyl group of the cysteine. In the polyisoprenylation step, a 15 carbon (trans, trans-farnesyl) or 20 carbon (all trans-geranylgeranyl) hydrocarbon group is covalently added to the protein.
The only reversible step in the process is the last step, methylation. Two enzymes mediate this final state of polyisoprenylated proteins. Polyisoprenylated protein methyl transferase (PPMTase), also known as isoprenyl carboxylmethyl transferase (ICMT), transfers a methyl group from S-adenosyl-L-methionine (SAM) to the C-terminal —COO− to form the methylated polyisoprenylated protein. PPMTase is essential to the developing embryo; knockout mice lacking PPMTase activity do not survive through mid-gestation. The second of the two enzymes is polyisoprenylated methylated protein methyl esterase (PMPMEase), which hydrolyzes the methyl esters of polyisoprenylated proteins to form the original proteins with free —COO− groups.
PPMTase and PMPMEase counterbalance the effects of each other. It is conceivable that the methylated and demethylated forms of prenylated proteins may be variously preferred for functional interactions by different protein targets, thus rendering PPMTase and PMPMEase very important moderators of polyisoprenylated protein function. Accordingly, manipulation of these enzymes should render significant effects on many cellular functions.
PMPMEase, through its possible regulation of the functions of various types of polyisoprenylated proteins, may exert profound effects on various intracellular events and consequently on animal physiology. Proteins such as the G-gamma subunits of heterotrimeric G-proteins of the G-protein coupled receptors, nuclear lamins, and guanine nucleotide-binding proteins such as Ras are polyisoprenylated and undergo methylation. These proteins mediate processes ranging from neurotransmitter signaling, cytoskeletal and intracellular transportation functions, cell proliferation, differentiation, and apoptosis. It could be inferred from this that aberrant levels of PMPMEase activity would be expressed through disease states such as cancers, neurodegenerative, and neuropsychiatric disorders. In fact, hyperactivity of monomeric G-proteins is implicated in an estimated 30% of cancers. Ghobrial I M, et al., Hematol. Oncol. Clin North Am. 2002, 16(5):1065-1088.
PMPMEase is inhibited by millimolar concentrations of the anticancer drugs tamoxifen and cyclophosphamide as well as by micromolar concentrations of the chemopreventive compound curcumin and polyunsaturated fatty acids such as arachidonic acid (AA). Prostaglandin (PG) A2 was 63-fold less potent than AA while PGE2 did not inhibit PMPMEase at 1 mM. AA's effects on cell death coupled with the expression of COX-2 in various tumors and tumor cell lines may imply that COX-2 converts AA into PGs, thereby destroying AA's ability to effectively inhibit PMPMEase and regulate cell growth. These results also show that balanced PMPMEase activity may be critical for normal cell viability.
It is an object of the invention to provide a method for detecting cancer which involves measuring the activity of the enzyme PMPMEase. While breast cancer is used as an exemplary embodiment herein, the invention is not limited to breast cancer.
The present invention is methods for the diagnosis, prediction, and treatment of cancer, based upon the activity of the enzyme PMPMEase.
PMPMEase activity is elevated in cancerous tissue compared to surrounding non cancerous tissue. Accordingly, an increase in PMPMEase activity is a marker for the presence of cancer, or a predisposition to cancer. Measurement of PMPMEase activity can therefore be the basis for cancer diagnosis, decisions on appropriate cancer treatment, awareness of a predisposition to cancer and potential cancer prevention, and monitoring of cancer therapy.
In one embodiment, the invention is a method of cancer diagnosis or prediction (together termed “detection”) including the steps 1) gathering a biological sample from a subject and 2) assaying the biological sample for PMPMEase activity. In a third step the PMPMEase activity in the biological sample is compared to PMPMEase activity in a control biological sample. In some preferred embodiments the biological sample is from a breast tumor. In other preferred embodiments, the biological sample is from lung, pancreatic, ovarian, prostate, colon, or liver cancerous tissue.
PMPMEase activity is elevated in cancerous tissue compared to surrounding non cancerous tissue. Accordingly, an increase in PMPMEase activity is a marker for the presence of cancer, or a predisposition to cancer.
In one embodiment, the invention is a method of cancer detection including the steps 1) gathering a biological sample from a subject and 2) assaying the biological sample for PMPMEase activity. In a third step the PMPMEase activity in the biological sample is compared to PMPMEase activity in a control biological sample.
Cancer as used herein means the presence of cells possessing characteristics typical of cancer causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Often, cancer cells will be in the form of a tumor, but such cells may exist alone within an animal, or may be a non-tumorigenic cancer cell, such as a leukemia cell. Cancers include, but are not limited to breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreas cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, a melanoma, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testis cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, and a chondrosarcoma. While the invention is particularly shown herein as applicable for detection of breast, lung, pancreatic, and ovarian cancers it is applicable to other cancers as well. In particular, it is likely that other cancers such as, but not limited to, melanoma, prostate, liver, colon, kidney, and brain cancers also have elevated levels of PMPMEase activity.
In another embodiment the invention is a method of preventing development or further development of cancer. If a subject is found to have elevated levels of PMPMEase activity, indicating a predisposition towards or presence of cancer, measures can be taken to prevent further development of the cancer. An appropriate cancer therapy depends upon the type and extent of the cancer and can be selected from chemotherapy, radiation therapy, surgery, antihormone therapy, receptor-targeted therapy, and immunotherapy. Other therapies include changes in diet and exercise. For example, in those cases where the PMPMEase activities are elevated, the consumption of foods rich in natural PMPMEase inhibitors such as polyunsaturated free fatty acids and curcumin may be recommended as an alternative remedy or for prevention of relapse. Specifically designed high affinity inhibitors of PMPMEase appropriately developed as drugs will be more suitable for targeting cancers with elevated PMPMEase activities.
In one preferred embodiment, the method is specific for detection of triple negative breast cancer. PMPMEase has particularly high activity in triple negative breast cancer. This is a particularly striking result since triple negative breast cancer has heretofore been difficult to treat. Although chemotherapy is a treatment option for triple negative breast cancer, the lack of a specific drug target implies that response to therapy is less than for the hormone-driven breast cancers which have more specific targeted therapeutic options and consequently better prognoses.
In another embodiment the invention is a method of screening foods, drugs, and other active agents for their ability to prevent or treat cancer by measuring their effect on PMPMEase activity.
In still another embodiment, the invention is the development of inhibitors targeted towards the suppression of PMPMEase activity to the level found in normal tissues.
In another embodiment, the invention is useful for treatment monitoring by measuring PMPMEase levels as a treatment progresses to determine its effectiveness.
PMPMEase activity can be measured in a number of ways that will be understood by those skilled in the art. Generally, enzyme activity is measured by measuring either the consumption of a substrate or the production of a product over time. For example, the biological sample can be incubated with a known PMPMEase substrate for a period of time, after which time the sample is assayed for the known enzymatic product.
The first step in the method is to collect the biological sample. This can be done through means well known in the art, by removing a sample of cells from the subject, but can also be accomplished by using previously isolated cells, or by performing the enzyme assay in vivo. The biological sample could alternatively be non-cellular biological material, such as non-cellular fractions of blood, saliva, or urine that can be used to measure PMPMEase activity. Preferred biological samples include tissue biopsies, scrapes (e.g. buccal scrapes), whole blood, plasma, serum, urine, saliva, cell culture, or cerebrospinal fluids. All of these are referred to herein as the biological sample or cells unless otherwise noted.
Often a control biological sample will also be obtained from the subject. This is a sample of biological material representative of healthy, cancer-free cells, preferably cells or tissue obtained from the same location of the subject. A control sample can also refer to an established level of PMPMEase activity representative of the cancer-free cells, which has been previously established based on measurements from normal, cancer-free cells.
The biological sample and control sample may need to be processed prior to the assay for PMPMEase activity. For example, it may be desirable to weigh and sonicate, homogenize or lyse the cells or tissues in a suitable buffer of pH at around 7.4 prior to incubation with the substrate. After incubation of the tissue extract/sample at or about 37° C. for a suitable amount of time, the reaction can be stopped by introducing conditions that denature proteins such as by heating or preferably adding a reagent that denatures the enzyme. Such reagents include, but are not limited to methanol and ethanol. Reagents such as acids or bases that may cause the ester bonds to break would generally be unsuitable. After centrifuging the stopped reactions to remove particulate matter, the supernatant liquids can then be analyzed.
The general PMPMEase reaction is shown as:
A number of PMPMEase substrates can be used to assay PMPMEase activity. In order for PMPMEase to be selective for the substrate and to avoid interference from other esterases, R2 is a polyisoprenyl group such as a trans, trans-farnesyl or all trans-geranylgeranyl group. Other hydrophobic groups can be used for R2 with varying degrees of selectivity for PMPMEase. R3 is naturally a methyl group as found in polyisoprenylated proteins and for the purposes of detecting the enzyme activity, a methyl as R3 will serve the purpose although ethyl and other usually hydrophobic groups will still be functional. R1 can be very diverse in chemical structure and application. For the purpose of sensitivity, groups that interact strongly with light such as fluorescent or chromophoric (light absorbing) groups will be most effective. The substrates can also be adapted for other detection methods such as radiolabeling.
A preferred substrate is L-N-(4-nitrobenzoyl)-S-trans, trans-farnesyl-cysteine methyl ester (RD-PNB). Other substrates that could be used are L-N-(2-nitrobenzoyl)-S-trans, trans-farnesyl-cysteine methyl ester, L-N-(3-nitrobenzoyl)-S-trans,trans-famesyl-cysteine methyl ester, L-N-(benzoyl)-S-trans, trans-farnesyl-cysteine methyl ester, L-N-(hippuryl)-S-trans, trans-farnesyl-cysteine methyl ester or L-N-(benzoyl-glycyl)-S-trans, trans-farnesyl-cysteine methyl ester (BzGFCM) and their geranyl and geranylgeranyl analogs. The structures of BzGFCM and RD-PNB are shown below.
Once the biological sample is obtained and processed, if desired, it is assayed for PMPMEase activity. The amount of PMPMEase activity in the tumor biological sample can be compared against the PMPMEase activity in normal control tissue. Preferably the difference between the increased level of PMPMEase activity in the biological sample and the level of PMPMEase activity in the control sample is statistically significant. Ethylenediaminetetraacetic acid (EDTA) can be included in the assay buffer to inactivate matrix metalloproteases.
In one embodiment, the amount of product formed is determined using reversed-phase high performance liquid chromatography with ultraviolet detection (HPLC-UV). The amounts of product formed will then be computed with the aid of a standard plot of amount of product against ultraviolet absorption to determine the relative activities of PMPMEase in the biological samples in order to make a diagnosis.
Other means for measuring the product formation or substrate depletion include radioactively labeling the substrate. In such as a scenario, reversed-phase HPLC analysis with radiochromatographic detection or other chromatographic methods coupled with radioactivity detection methods can be used.
An increase in an enzyme's activity may be due to increased levels of the protein, a mutation that makes the protein more active (also called gain-of-function mutation to distinguish it from loss-of-function mutations that result in a less active protein), or the loss of a moderating factor such as a natural endogenous or exogenous inhibitor. Increased expression of enzyme can be monitored by quantifying the levels of messenger RNA (mRNA) using RT-PCR (less accurate) or levels of protein using ELISA and/or western blotting (WB) and/or immunohistochemistry (IH). ELISA, WB and IH— which involve the use of antibodies—can be jointly termed immunological methods and are more predictive of increased protein levels and/or activity than RT-PCR. Changes in protein activity that are due to mutations only but which do not affect gene regulation i.e. do not cause changes in mRNA and therefore protein synthesis cannot be detected by western blotting and ELISA. Loss of a moderating factor is also not detectable by WB and ELISA using antibodies directed at PMPMEase.
Changes in PMPMEase's activity due to any of the three factors above can be detected by the above discussed PMPMEase enzymatic assays. However, for some, perhaps a significant proportion of the cases, the increase in PMPMEase enzyme activity will be due to an increase in PMPMEase expression. Therefore, RT-PCR and the immunological methods for measuring increase in PMPMEase levels can also be used to predict increased PMPMEase activity. Because of the reasons stated above and the fact that proteins may be inactive due to denaturation and/or functional regulation, the true physiological impact of a protein can be most accurately evaluated using a functional assay of its activity.
The examples below serve to further illustrate the invention, to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices, and/or methods claimed herein are made and evaluated, and are not intended to limit the scope of the invention. In the examples, unless expressly stated otherwise, amounts and percentages are by weight, temperature is in degrees Celsius or is at ambient temperature, and pressure is at or near atmospheric.
Materials and Methods
Ten breast cancer tumor samples together with respective matching adjacent normal tissues were obtained from ProteoGenex (Culver City, Calif.). The specimens were from ten Caucasian women ranging from age 44 to 77 years. The ER, HER2 and progesterone receptor status for three of the ten samples was known. The sample BrC5 was triple negative, BrC7 was positive for estrogen and progesterone receptors and negative for HER2, and BrC10 was positive for HER2 only. The tissues were collected and surgically excised by certified medical pathologists at ProteoGenex and snap-frozen in liquid nitrogen within 30 min of surgery. All the samples were of stage IIIA with histological diagnoses of infiltrating ductal carcinomas. The samples and their characteristics are listed in Table 1.
The TNM Classification of Malignant Tumours (TNM) is a cancer staging system that describes the extent of cancer in a patient's body. T describes the size of the tumor and whether it has invaded nearby tissue and ranges from 0 to 4, N describes the degree of spread to regional lymph nodes and ranges from 0 to 3, and M describes presence of metastasis and is either 0 (no metastasis) or 1 (metastasis beyond regional lymph nodes).
The substrate L-N-(4-nitrobenzoyl)-S-trans, trans-farnesyl-cysteine methyl ester (RD-PNB) was synthesized as described in Lamango, N. S. et al. (2009) TOEIJ 2, 12-27(1). This substrate is an adaptation of the PMPMEase substrates described in Lamango, N. S. (2005) J Biochem Mol Toxicol 19, 347-357 and Oboh, O. T. et al. (2008) J Biochem Mol Toxicol 22, 51-62.
The frozen tumor and adjacent matching controls were weighed and sonicated in ice-cold 100 mM Tris-HCl buffer, pH 7.4 containing 1 mM ethylenediaminetetraacetic acid (EDTA) (5× mass of tissue). Aliquots of the homogenates (95 μL) were incubated with the RD-PNB substrate (1 mM) at 37° C. for 3 h. The reactions were stopped by the addition of 200 μL methanol. After chilling them at −20° C. for at least 5 min, they were centrifuged at 5000×g for 5 min and the supernatants analyzed by reversed-phase HPLC with UV-detection at 260 nm as described in Lamango, N. S. et al. (2009) TOEIJ 2, 12-27(1). The amount of product formed was determined using a calibration plot of HPLC UV-peak areas for known amounts of product standards. The total protein in the homogenates was determined using the bicinchoninic acid method.
Results
When the samples were analyzed based on the weighed mass of tissue rather than the total protein (
Specific Samples
BrC5, known to be triple negative, shows the most enhanced PMPMEase activity when based on the weighed mass of tissue (
In a time-course analysis of substrate hydrolysis by BrC5 control and tumor tissues, 10 μL of homogenate was used instead of the 95 μL used in the initial screening of the samples. Briefly, 10 μL the homogenate was incubated with the RD-PNB substrate (1 mM) at 37° C. At the indicated time points, the reactions were stopped by the addition of 200 μL methanol. After chilling them at −20° C. for at least 5 min, they were centrifuged at 5000×g for 5 min and the supernatants analyzed by reversed-phase HPLC with UV-detection at 260 nm as described in Lamango, N. S. et al. (2009) TOEIJ 2, 12-27(1). The amounts of product formed were determined using a calibration plot of HPLC UV-peak areas for known amounts of product standards. As shown in
Human tissue microarrays (TMAs) were supplied by and immunohistochemistry was conducted at US Biomax (Rockville, Md.). Two breast cancer TMAs consisted of 150 and 100 cores involving 75 and 50 cases. All the tissues were formalin-fixed, paraffin-embedded and mounted on positively charged SuperFrost Plus glass slides. The sections were all 5 μm thick and 1 or 1.5 μm in diameter. The cores were separated from each other by 0.25 mm.
Rabbit polyclonal antibody directed against PMPMEase (human carboxylesterase 1, hCE1) was obtained from Santa Cruz Biotechnology (Santa Cruz, Calif.). ImmPRESS Reagent anti-Rabbit Ig (peroxidase) reagent MP7401 and ImmPRESS Reagent anti-Rabbit Ig (peroxidase) reagent MP7405 were purchased from Vector Laboratories. DAB (DAKO Cytomation) was used as substrate chromogen. Antigen retrieval solution was purchased from DakoCytomation (Target Retrieval solution). The Antigen retrieval was performed before incubating with primary antibody.
The sections were deparaffinized in xylene and hydrated through a gradient ethanol series. These were rinsed in water for 5 min. These were then incubated in 3% hydrogen peroxide for 5 min to block endogenous peroxidase activity followed by two 5 min washes in water. The antigens were retrieved by simmering the sections in a microwave for 20 min in 1× antigen retrieval solution. These were allowed to cool to room temperature over a 15 min period before washing with three 5 min washes in PBST buffer. The sections were then incubated for 30 min with ready-to-use (2.5%) normal horse blocking serum. They were then incubated with diluted PMPMEase antibody (0.25 or 0.20 μg/mL). The slides were rinsed for three 5 min periods in buffer, incubated for 30 min in ImmPRESS reagent followed by a further 3×5 min washes in buffer. They were then exposed to the peroxidase substrate DAB solution. The sections were rinsed in tap water and counterstained in Hematoxylin QS (Vector Labs). These were then mounted using permanent mounting medium from Sigma (St. Louis, Mo.). The DAB substrate-chromogen stains the target antigen site dark brown while the hematoxylin stains the nuclei blue. The IHC-stained slides were scanned at 20× magnification.
Immunohistochemistry Scoring
The method used to score the PMPMEase immunoreactivity was adapted from that of Bremnes et al. (2002). The intensity of the staining was given a score of 0 (no staining), 1 (trace), 2 (weak), 3 (intermediate), 4 (strong), or 5 (very strong). The score of the staining intensity was then multiplied by the percentage of the immunoreactive tumor cells. The overall scores ranged between 0 and 500 with those between 1 and 100 described as trace, 101 to 200 as weak, 201 to 300 as intermediate, 301 to 400 as strong, and 401 to 500 as very strong. The evaluation and scoring was conducted without prior knowledge of the diagnosis of the individual samples on the TMAs.
Results
The immunological results show consistent immunoreactivity of PMPMEase in the tumors as opposed to the normal tissues or normal adjacent tissues (NAT). Although there was significant inter- and intra-slide variability in the intensities of the brown staining, which is indicative of the presence of PMPMEase, the staining was consistently more in tumor samples than in surrounding non-tumor tissues.
Specific subtypes of breast cancer were scored and graphed selectively. The results are shown in
Lung cancer screening was performed following the above procedures. Two lung cancer TMAs were used consisting of a total of 416 cores involving 416 separate cases.
Relative PMPMEase immunoreactivities were measured for various types of pancreatic cancer. TMAs were made from 208 cores of 101 and 104 cases and tested as described above and the results are shown in
Relative PMPMEase immunoreactivities were measured for various types of ovarian cancer. TMAs were made from 208 cores of 101 and 104 cases. PMPMEase immunoreactivities were scored as described above and the results are shown in
Relative PMPMEase immunoreactivities were measured for prostate cancer. TMAs were made from 113 cores of 113 cases. PMPMEase immunoreactivities were scored as described above and the results are shown in
Relative PMPMEase immunoreactivities were measured for adenocarcinoma (AC) and mucinous adenocarcinoma (MAC) of the colon. TMAs were made from 204 cores of 204 cases. PMPMEase immunoreactivities were scored as described above and the results are shown in
Relative PMPMEase immunoreactivities were measured for hepatocellular carcinoma (HCC) and bile duct adenocarcinoma (AC). TMAs were made from 203 cores of 203 cases. PMPMEase immunoreactivities were scored as described above and the results are shown in
Normal liver tissues show very strong immunoreactivity for PMPMEase that is disrupted by the regular pattern of canaliculi. Unlike the normal liver tissues, HCC lacks the canaliculi and depending on the specific cases, PMPMEase immunoreactivity may range from weak to very strong (see error bars in HCC and bile duct AC relative to normal). PMPMEase in the liver tumors can serve as a companion diagnostic method to determine whether the drugs targeting PMPMEase for inhibition are likely to result in therapeutic benefit.
Modifications and variations of the present invention will be apparent to those skilled in the art from the forgoing detailed description. All modifications and variations are intended to be encompassed by the following claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.
This invention was made with government support under NIH/NIGMS/SCORE grant GM 08111-35 and Pharmaceutical Research Center NIH/NCRR grant G12 RR0 3020. The government has certain rights in the invention.
