WO2011051938A1

WO2011051938A1 - Composition for treatment of thyroid cancer with fts and analogs thereof

Info

Publication number: WO2011051938A1
Application number: PCT/IL2010/000883
Authority: WO
Inventors: Yoel Kloog; Ran Levi
Original assignee: Ramot At Tel-Aviv University Ltd.
Priority date: 2009-10-26
Filing date: 2010-10-26
Publication date: 2011-05-05
Also published as: JP2013508450A

Abstract

Disclosed are pharmaceutical compositions and methods of treating thyroid cancer characterized by elevated levels of K- Ras relative to a normal thyroid cell. The method entails administering to a thyroid cancer patient a pharmaceutical composition that includes a therapeutically effective amount of FTS (farnesylthiosalicylic acid or Salirasib) or an FTS analog.

Description

COMPOSITION FOR TREATMENT OF THYROID CANCER WITH FTS AND

ANALOGS THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the filing date of United States Provisional Patent Application No. 61/254,879, filed October 26, 2009, the disclosure of which is hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Follicular thyroid carcinoma is the most common endocrine malignancy [Hundahl, et al . , Cancer 83 (12) : 2638-48

(1998) ; Perkin, et al., CA Cancer J. Clin. 55 (2) : 74-108 (2005) ] . More than 95% of thyroid carcinomas are derived from follicular cells. Most carcinomas that are derived from follicular epithelial cells are indolent tumors that can be effectively managed by surgery with or without radioactive- iodine ablation. However, certain subsets of these tumors can behave aggressively, and there is currently no effective form of treatment [Sherman, S.I., Lancet 361 (9356) : 501-11 (2003); Schlumberger, M.J., New England Journal of Medicine 338 (5) :297-306 (1998)].

[0003] Follicular thyroid carcinoma compromises a broad spectrum of tumors ranging from well-differentiated to undifferentiated types, on the basis of histological and clinical parameters [Ros, et al. , Biochimie 81 (4) : 389-96

(1999) ; Hunt, et al., Am. J. Surg. Pathol. 27 (12) : 1559-64 (2003) ] . Well-differentiated thyroid carcinoma includes papillary (PTC) and follicular (FTC) types. They have a generally good prognosis. In contrast, undifferentiated or anaplastic thyroid carcinoma (ATC) is highly aggressive and has a very poor prognosis [Chiacchio, et al., Minerva J. Endocrinol. 33 (4) : 341-57 (2008); Sipos, et al., Expert Opin. Pharmacother 9 (15) : 2627-37 (2008)]. ATC results in a rapidly enlarging neck mass that invades adjacent tissues and metastasizes to different parts of the body, particularly into the bones. There is currently no effective treatment for anaplastic thyroid carcinoma. Surgery, chemotherapy and radiotherapy are the conventional therapeutic strategies performed in the attempt to improve survival. Surgery is not feasible in many patients, with operability varying from 17-65% across reported series [Ahuja, et al., J. e ndocrinol. Invest. 10 (3) : 303-10 (1987)]. Due to the aggressive nature and potential for systemic spread of ATC, many different chemotherapy regimens have been tried, including doxorubicin, which has shown at best a 22% partial response rate. Survival is usually within 1 year of diagnosis [Kebebew, et al., Cancer 103 (7) : 1330-5 (2005).

BRIEF SUMMARY OF THE INVENTION

[0004] A first aspect of the present invention is directed to a method for treating a patient with a thyroid cancer characterized by elevated levels of K-Ras relative to a normal thyroid cell. The method entails administering to the patient a composition that includes a therapeutically effective amount of farnesylthiosalicylic acid (also referred to herein as FTS or Salirasib) or an FTS analog, which together are defined by the formula described herein, and a pharmaceutically acceptable carrier. Compositions for use in practicing these methods, as well as methods of making them, are also provided.

[0005] The present inventors have discovered that thyroid cancers characterized by elevated K-Ras levels, such as papillary thyroid carcinoma, follicular thyroid carcinoma, and anaplastic thyroid carcinoma, can be treated with various Ras antagonists such as S-trans , trans-farnesylthiosalicylic acid and its analogs. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Fig. 1A and B are bar graphs showing that the Ras inhibitor FTS inhibits growth of thyroid follicular cells with high levels of Gal-3 protein, wherein Fig. 1A shows reduction in cell proliferation by FTS on thyroid carcinomas ARO, MRO, and NPA (and lack of any such effect on TT cells) (15000 cells/96-wells plate) grown in the presence of a relatively low serum concentration (5%) and treated with 50, 75 and ΙΟΟμΜ FTS or 0.1% DMSO (control) for 24 hours, wherein cell proliferation was then determined by incorporation of BrdU into the DNA, and wherein data are presented as the percent of BrdU in the FTS-treated cells relative to the control; and Fig. IB shows viabilty of FTS-treated thyroid carcinoma cells grown and treated as described above then subjected to determination of determination of live cells using the AlamarBlue regent, wherein data are presented as the percent of AlamarBlue fluorescence in the FTS-treated cells relative to the control (means ± SD, n = 3) .

[0007] Fig. 2 is an immunoblot that shows levels of active Ras, ERK and Gal-3 in the thyroid carcinoma cell lines ARO, MRO, NPA and TT cells, that were homogenized, followed by subjecting aliquots of the homogenates to the determination of levels of Gal-3, Ras, Ras-GTP, Ras-GTP isoforms (N-Ras and K-Ras) and the Ras downstream effector phospho-ERK using SDS- PAGE and immunoblotting with specific antibodies, wherein β- tubulin served as a loading control, and wherein immunoblots were visualized by ECL.

[0008] Figs. 3A, B and C are bar graphs showing that FTS inhibits Ras and its signals in various thyroid cancer cell lines, wherein Fig. 3A shows that FTS reduces the level of total Ras.GTP in ARO, MRO and NPA cells, wherein the cells were plated as described in Fig. 1A then treated with 75μΜ FTS or with the vehicle control for 48 hours, lysed and subjected to quantification of active Ras.GTP and total Ras followed by immunoblotting with pan Ras Ab; Fig. 3B shows that FTS reduces the level of K-Ras.GTP in ARO, MRO and NPA cells grown as described for Fig. 3A but under conditions of relatively low serum condition (0.5%) and then treated with ΙΟμΜ FTS or with the vehicle control for 24 hours, followed by lysis and then quantification of active K-Ras.GTP and for total K-Ras followed by immunoblotting with specific anti K-Ras antibody; and wherein Fig. 3C shows that FTS reduces the levels of phospho-ERK in ARO and MRO cells treated with 75μΜ FTS for 48 hours then lysed and subjected to immunobloting with anti ERK and anti phosphor-ERK Ab.

[0009] Figs 4A and A' , B, C, and D and D' are bar graphs showing that FTS treatment increases the levels of P21 and Ttf-1 in thyroid carcinoma cells; wherein Figs. 4A and 4A' show that FTS induces upregulation of p21 and Ttfl respectively in thyroid carcinoma cells ARO, MRO and NPA cells that were plated and treated with 75μΜ FTS as described in Fig 3A, followed by lysis and SDS-PAGE and immunoblotting with anti-p21and anti Ttf-1 or anti tubulin (control) antibodies, wherein the levels of p21 in the FTS-treated ARO and MRO and NPA cell were higher than in the corresponding controls (*P< 0.05, **p<0.01), and the levels of Ttf-1 in the FTS-treated ARO and MRO cells were also higher than in the corresponding controls (*P≤ 0.05), but wherein not much difference was recorded in NPA and TT cells; Fig. 4B shows that dominant negative Ras increases the levels of Ttfl in thyroid carcinoma cells ARO and MRO transfected with vectors expressing the dominant negative GFP-Ras 17N or GFP (control) followed by lysis 48h after transfection and immunblotting with anti-Ttf-1 and anti β-tubulin Ab; Fig. 4C shows statistical analysis of confocal fluorescence images of control ARO cells and of cell treated with FTS, wherein the cells (2xl0⁵ cells) were plated onto glass cover slips then treated 48 h with 75μΜ FTS or with the vehicle control and labeled with Hoechst and with rabbit anti-Ttfl Ab followed by fluoresceine-labeled goat anti-rabbit Ab and imaged; and Figs. 4D and 4D' show that the MEK inhibitor U0126 induces increase in p21 and Ttfl respectively in ARO and MRO cells plated at a density of 2xl0⁵ cells/ 6-cm plate and grown 24 h in the RPMI/5% FCS with and without ΙΟμΜ U0126, followed by lysis, SDS-PAGE and then immunobloting with anti-Ttf-1 anti-p21 and anti- -tubulin Ab.

[0010] Fig. 5 is a bar graph showing that FTS disrupts K-Ras-Gal-3 colocalization in the cell membrane of ARO cells

(2xl0⁵ cells) that were plated onto glass cover slips and grown in the presence of 0.1% DMSO (control) or in the presence 75μΜ FTS for 72 hours then labeled with mouse pan anti Ras Ab followed by cy3-labeled donkey anti-mouse Ab and with rat anti-Gal-3 Ab followed by fluoresceine-labeled goat anti-rat Ab.

[0011] Figs. 6A, B and C are graphs showing that FTS inhibits ARO cell tumor growth in a nude mouse model, wherein ARO cells were injected s.c. into the flanks of nude mice, followed by daily treatment with oral FTS (60 mg/kg, 10 mice) or with the vehicle control (10 mice) begun 7 days after cell implantation; wherein Fig. 6A shows volumes of ARO cell tumors in control and FTS-treated mice as a function of time (means ± SE n=10 * P<0.05, control vs. FTS); and Fig. 6B shows weights of ARO cell tumors in control mice and in FTS-treated mice, wherein tumor weights were determined on day 25 of the treatment (means ± SE, n= 10, ** P < 0.01); and wherein Fig. 6C shows activity of Gal-3, Ras, phosph-ERK and β-tubulin assayed by immunoblotting (in terms of apparent levels of densitometric analysis of Gal-3, Ras, Ras-GTP and phospho- ERK expression, which were normalized by β-tubulin expression) (mean + SE, n=4, * P<0.05, control vs. FTS). DETAILED DESCRIPTION

[0012] The Ras Antagonists

[0013] Ras proteins e.g., H-, N- and K-Ras, act as on-off switches that regulate signal-transduction pathways controlling cell growth, differentiation, and survival.

[Reuther, et al., Curr. Opin. Cell Biol. 12:157-65 (2000)]. They are anchored to the inner leaflet of the plasma membrane, where activation of cell-surface receptors, such as receptor tyrosine kinase, induces the exchange of guanosine diphosphate

(GDP) for guanosine triphosphate (GTP) on Ras and the conversion of inactive Ras-GDP to active Ras-GTP.

[Scheffzek, et al. Science 277:333-7 (1997)]. Termination of these signals involves hydrolysis of the Ras-GTP to Ras-GDP.

[Scheffzek, et al., Science 277:333-338 (1997).] Besides the cell-proliferation promotion by wild-type Ras, several mutated forms of Ras are defective in their GTP hydrolysis liability and are therefore constitutively active. [Barbacid, Biochem. 56:779-827 (1987); Box, Eur. J. Cancer 32:1051-1054 (1995).] These oncogenic Ras proteins, which are found in many cancer types, contribute to malignancy and are therefore considered favored targets for directed therapy. [Bos, Cancer Res. 49:4682-4689 (1989).] The active Ras protein promotes oncogenesis through activation of multiple Ras effectors that contribute to deregulated cell growth, differentiation, and increased survival, migration and invasion. [See, e.g., Downward, J., Nat. Rev. Cancer 3:11-22 (2003); Shields, J. M. , et al., Trends Cell Biol. 20:147-541 (2000); and Mitin, N . , et al., Curr. Biol. 25:R563-74 (2005)].]

[0014] Association of Ras to the plasma membrane has been shown to be crucial for its activity in both the wild type and the mutated constitutively active forms. [Boguski, et al., Nature, 366:643-654 (1993); Cox, et al., Curr. Opin. Cell Biol. 4:1008-1016 (1992); Marshall, Curr. Opin. Cell Biol. 8:197-204 (1996).] At least two structural elements are required for this association; the first is a farnesylcysteine carboxy methyl ester at the carboxy terminal of Ras, and the second element resides at the adjacent upstream sequence and varies among different Ras isoforms. [Hancock, et al., EMBO J. 10:4033-4039 (1991); Hancock, et al., Cell 57:1167-1177 (1989).] Normal Ras activity requires specifically the farnesyl isoprenoid moiety [Cox, et al., Curr. Opin. Cell Biol. 4:1008-1016, (1992); Cox, et al., Mol. Cell. Biol. 12:2606-2615 (1992)] which acts as a specific recognition unit to allow binding of H-Ras with galectin-1 [Elad-Sfadia, et al., J. Biol. Chem. 277:37169-37175 (2002), Rotblat, et al., J. Biol. Chem. 54:3112-3118 (2004)] and K-Ras with galectin-3 [Elad-Sfadia, et al., J. Biol. Chem. 279:34922-34930 (2004)] promoting strong membrane association and robust signaling.

[0015] FTS is known as a Ras inhibitor that acts in a rather specific manner on the active, GTP-bound forms of H-, N-, and K- Ras proteins. [Weisz, B., et al., Oncogene 18:2579-2588 (1999); Gana-Weisz, M.,et al., Clin. Cancer Res. 8:555-65 (2002)]. More specifically, FTS competes with Ras-GTP for binding to specific saturable binding sites in the plasma membrane, resulting in mislocalization of active Ras and facilitating Ras degradation. [Haklai, et al., Biochemistry 37 (5) : 1306-14 (1998)]. This competitive inhibition prevents active Ras from interacting with its prominent downstream effectors and results in reversal of the transformed phenotype in transformed cells that harbor activated Ras. As a consequence, Ras-dependent cell growth and transforming activities, both in vitro and in vivo, are strongly inhibited by FTS. [Weisz, B., et al., supra.; Gana- Weisz, M., et al., supra.]. [0016] Ras antagonists useful in the present invention include FTS and its structural analogs, are described below.

[0017] The Ras antagonists are represented by the formula:

wherein represents wherein R¹ represents farnesyl, or geranyl- geranyl; R² is COOR⁷, CONR⁷R⁸, or COOCHR⁹OR¹⁰, wherein R⁷ and R⁸ are each independently hydrogen, alkyl, or alkenyl, including linear and branched alkyl or alkenyl, which in some embodiments includes C1-C4 alkyl or alkenyl; wherein R⁹ represents H or alkyl; and wherein R¹⁰ represents alkyl, including linear and branched alkyl and which in some embodiments represents C1-C4 alkyl; and wherein R³, R⁴, R⁵ and R⁶ are each independently hydrogen, alkyl, alkenyl, alkoxy (including linear and branched alkyl, alkenyl or alkoxy and which in some embodiments represents C1-C4 alkyl, alkenyl or alkoxy) , halo, trifluoromethyl , trifluoromethoxy, or alkylmercapto; and wherein X represents S. In embodiments wherein any of R⁷, R⁸, R⁹ and R¹⁰ represents alkyl, it is preferably methyl or ethyl.

[0018] In some embodiments, the Ras antagonist is S- trans, trans-farnesylthiosalicylic acid or FTS (wherein R¹ is farnesyl, R² is COOR⁷, and R⁷ is hydrogen) .

[0019] In some embodiments, the FTS analog is halogenated, e.g., 5-chloro-FTS (wherein R¹ is farnesyl, R² is COOR⁷, R⁴ is chloro, and R⁷ is hydrogen) , and 5-fluoro-FTS (wherein R¹ is farnesyl, R² is COOR⁷, R⁴ is fluoro, and R⁷ is hydrogen) .

[0020] In other embodiments, the FTS analog is FTS-methyl ester (wherein R¹ represents farnesyl, R² represents COOR⁷, and R⁷ represents methyl), FTS-amide (wherein R¹ represents farnesyl, R² represents CONR⁷R⁸, and R⁷ and R⁸ both represent hydrogen) ; FTS-methylamide (wherein R¹ represents farnesyl, R² represents CONR⁷R⁸, R⁷ represents hydrogen and R⁸ represents methyl) ; and FTS-dimethylamide (wherein R¹ represents farnesyl, R² represents CONR⁷R⁸, and R⁷ and R⁸ each represents methyl) .

[0021] In yet other embodiments, the Ras antagonist is an alkoxyalkyl S-prenylthiosalicylate or an FTS-alkoxyalkyl ester (wherein R² represents COOCHR⁹OR¹⁰) . Representative examples include methoxymethyl S-farnesylthiosalicylate (wherein R¹ is farnesyl, R⁹ is H, and R¹⁰ is methyl) ; methoxymethyl S- geranylgeranylthiosalicylate (wherein R¹ is geranylgeranyl, R⁹ is H, and R¹⁰ is methyl) ; methoxymethyl 5-fluoro-S- farnesylthiosalicylate (wherein R¹ is farnesyl, R⁵ is fluoro, R⁹ is H, and R¹⁰ is methyl) ; and ethoxymethyl S- farnesylthiosalicyate (wherein R¹ is farnesyl, R⁹ is methyl and R¹⁰ is ethyl) . In each of the embodiments described above, unless otherwise specifically indicated, each of R³, R⁴, R⁵ and R⁶ represents hydrogen.

[0022] Compositions and Methods

[0023] The terms "effective amount", "therapeutically effective amount" or "pharmaceutically effective amount" as used herein, refer to a sufficient amount of the Ras antagonist that will ameliorate at least one symptom of the thyroid cancer and its associated manifestations, diminish extent or severity of the disease, delay or retard disease progression, achieve partial or complete remission, prolong survival and combinations thereof. Appropriate "effective" amounts for any cancer patient can be determined using techniques, such as a dose escalation study. Specific dose levels for any particular patient will depend on several factors such as the potency of the Ras antagonist, the age, weight, and general health of the patient, and the severity of the cancer. The average daily dose of the Ras antagonists of the present invention generally ranges from about 200 mg to about 2000 mg, in some embodiments from about 400 to about 1600 mg, and some other embodiments from about 600 to about 1200 mg, and in yet other embodiments, from about 800 mg to about 1200 mg.

[0024] The terms "administer," "administering",

"administration," and the like, as used herein, refer to the methods that may be used to enable delivery of the Ras antagonist to the desired site of biological action. Medically acceptable administration techniques suitable for use in the present invention are known in the art. See, e.g., Goodman and Gilman, The Pharmacological Basis of Therapeutics, current ed. ; Pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, Pa. In some embodiments, the Ras antagonist is administered orally. In other embodiments, the Ras antagonist is administered parenterally (which for purposes of the present invention, includes intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular and infusion) . Other administration routes such as topical and rectal administration may also be suitable.

[0025] The term "pharmaceutically acceptable" as used herein, refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic.

[0026] The term "pharmaceutical composition, " as used herein, refers to the Ras antagonist, optionally combined

(e.g., mixed) with a pharmaceutically acceptable carrier. These ingredients are non-toxic, physiologically inert and do not adversely interact with the Ras antagonist (and any other active agent (s) that may be present in the composition). Carriers facilitate formulation and/or administration of the active agents. Pharmaceutical compositions of the present invention may further contain one of more excipients. [0027] Oral compositions for the Ras antagonist can be prepared by bringing the agent (s) into association with (e.g., mixing with) the carrier, the selection of which is based on the mode of administration. Carriers are generally solid or liquid. In some cases, compositions may contain solid and liquid carriers. Compositions suitable for oral administration that contain the active are preferably in solid dosage forms such as tablets (e.g., including film-coated, sugar-coated, controlled or sustained release) , capsules, e.g., hard gelatin capsules (including controlled or sustained release) and soft gelatin capsules, powders and granules. The compositions, however, may be contained in other carriers that enable administration to a patient in other oral forms, e.g., a liquid or gel. Regardless of the form, the composition is divided into individual or combined doses containing predetermined quantities of the Ras antagonist.

[0028] Oral dosage forms may be prepared by mixing the Ras antagonist, typically in the form of an active pharmaceutical ingredient with one or more appropriate carriers (optionally with one or more other pharmaceutically acceptable excipients) , and then formulating the composition into the desired dosage form e.g., compressing the composition into a tablet or filling the composition into a capsule or a pouch. Typical carriers and excipients include bulking agents or diluents, binders, buffers or pH adjusting agents, disintegrants (including crosslinked and super disintegrants such as croscarmellose) , glidants, and/or lubricants, including lactose, starch, mannitol, microcrystalline cellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, dibasic calcium phosphate, acacia, gelatin, stearic acid, magnesium stearate, corn oil, vegetable oils, and polyethylene glycols. Coating agents such as sugar, shellac, and synthetic polymers may be employed, as well as colorants and preservatives. See, Remington 's

Pharmaceutical Sciences, The Science and Practice of Pharmacy, 20th Edition, (2000) .

[0029] Liquid form compositions include, for example, solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The active agent (s), for example, can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent (and mixtures thereof), and/or pharmaceutically acceptable oils or fats. Examples of liquid carriers for oral administration include water (particularly containing additives as above, e.g., cellulose derivatives, preferably in suspension in sodium carboxymethyl cellulose solution) , alcohols (including monohydric alcohols (including monohydric alcohols and polyhydric alcohols, e.g., glycerin and non-toxic glycols) and their derivatives, and oils (e.g., fractionated coconut oil and arachis oil) . The liquid composition can contain other suitable pharmaceutical excipients such as solubilizers , emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colorants, viscosity regulators, stabilizers and osmoregulators.

[0030] Carriers suitable for preparation of compositions for parenteral administration include Sterile Water for Injection, Bacteriostatic Water for Injection, Sodium Chloride Injection (0.45%, 0.9%), Dextrose Injection (2.5%, 5%, 10%), Lactated Ringer's Injection, and the like. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, and in oils. Compositions may also contain tonicity agents (e.g., sodium chloride and mannitol) , antioxidants (e.g., sodium bisulfite, sodium metabisulfite and ascorbic acid) and preservatives (e.g., benzyl alcohol, methyl paraben, propyl paraben and combinations of methyl and propyl parabens) . [0031] The thyroid cancers amenable to treatment in accordance with the methods of the present invention are characterized by elevated levels of K-Ras relative to a normal thyroid cell, which can be determined by comparison of immunoblots of thyroid cancer cells with those of normal thyroid cells. Such cancers include papillary thyroid carcinoma, follicular thyroid carcinoma, and anaplastic thyroid carcinoma.

[0032] The pharmaceutical composition containing the Ras antagonist may be packaged and sold in the form of a kit. For example, the composition might be in the form of one or more oral dosage forms such as tablets or capsules. The kit may also contain written instructions for carrying out the inventive methods as described herein.

[0033] In some embodiments, the Ras antagonist is administered by dosing orally on a daily basis (in single or divided doses) for three weeks, followed by a one-week "off period", and repeating until remission is achieved.

[0034] The Ras antagonist may be used alone or in conjunction with other treatment agents such as biological anti-cancer agents (e.g., antibodies), chemotherapeutic agents and radiation, as a front-line treatment strategy (e.g., as a first course of treatment in a newly diagnosed cancer patient, and whether or not the cancer has metastasized) or as a second-line treatment strategy (e.g., treatment of a cancer patient who has been previously treated using at least one other agent but has not responded to the previous agent (s) or has developed a resistance thereto, which may have resulted in termination of the therapy even before it could achieve an appreciable therapeutic efficacy) .

[0035] The invention will now be described in the following non-limiting working examples.

[0036] Example 1 — In Vitro Experiments

[0037] Cell lines and reagents [0038] The human follicular thyroid cancerous cell lines ARO and MRO, and the human anaplastic thyroid cancer cell line NPA were a gift from Zaki Kraiem from the Endocrinology Institute, Soarsky Medical Center Tel Aviv. The Medullary thyroid carcinoma cell line TT was purchased from ATCC

(catalog number: CRL-1803™) . All cell lines were cultured in RPMI medium containing 10% fetal calf serum (FCS) , 2 mM L- glutamine, 100 U/ml penicillin andlOO g/ml streptomycin. The cells were incubated at 37 °C in a humidified atmosphere with 5% C0₂.

[0039] FTS was a gift from Concordia Pharmaceuticals (Ft. Lauderdale, FL) . The ECL kit was purchased from Amersham (Arlington Heights, IL) ; Hoechst 33258 from Sigma-Aldrich (St. Louis, MO) . U0126 was from AG Scientific (San Diego, CA) . Mouse anti-pan-Ras (Ab-3) , mouse anti-N-Ras and mouse anti-K-Ras antibodies were obtained from Calbiochem; rabbit anti-p21, rabbit anti-Ttfl and rabbit anti β-Tubulin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA) ; mouse anti-phospho-ERK were from Sigma-Aldrich; rabbit anti-phospho-Akt (ser473) and rabbit anti-GAPDH (14C10) antibodies were from Cell Signaling Technology (Beverly, MA) . Peroxidase-goat anti-mouse IgG, peroxidase-goat anti-rat IgG, and peroxidase-goat anti-rabbit IgG were from Jackson ImmunoResearch Laboratories (West Grove, PA) .

[0041] Transfection

[0042] For the transfection assay, 2 x 10⁵ ARO and MRO cells/well were plated in 6-well plates. The following day, the cells were transfected with plasmid DNA coding for GFP-Rasl7N (2 pg) or with a vector with control coding for green fluorescent protein (2 pg) using Lipofetamine™ 2000 transfection kit (cat no. 11668-027, Invitrogen) according to manufacturer's instructions. [0043] Cell viability

[0044] ARO, MRO, NPA and TT cells (1.5 x 10⁴ cells/well in 96-well plates) were treated with 50, 75 and 100 μΜ FTS or the vehicle (0.1% DMSO) for 24 h. Cell viability was estimated by using AlamarBlue assay according to manufacturer's instructions (Serotec, Oxford, UK) .

[0045] Cell proliferation assay

[0046] ARO, MRO, NPA and TT cells were plated in 5% FCS media at a density of 1.5 x 10⁴ cells/well in 96-well plates. The next day, cells were treated with 50, 75, or 100 μΜ FTS or the vehicle (0.1% DMSO). Proliferation was assessed by incorporation of 5-bromo-2-deoxyuridine (BrdU) , using the BrdU cell-proliferation assay kit (Calbiochem) .

[0047] MTT assay

[0048] Cell proliferation was determined after 6 days by using the 3- ( 4 , 5-dimethylthiazol-2-yl ) -2 , 5-diphenyltetrazolium bromide (MTT) assay, which determines mitochondrial activity in living cells. The cells were incubated with 0.1 mg/mL MTT for 2 hours at 37 °C then lysed with 100% Me₂S0₄. Results were quantified by reading the absorbance at 570 to 630 nm.

[0049] Western Immunoblotting

[0050] ARO, MRO, NPA (0.4 x 10⁶ cells/10-cm) and TT cells (0.5 x 10⁶ cells/iriL) were cultured in RPMI 1640 medium containing 5% FCS. Cells were treated with 75 μΜ FTS or with the vehicle (0.1% DMSO) for 48 h then lysed and subjected to SDS PAGE and immunoblot analysis as detailed earlier [Elad-Sfadia, et al._r J. Bol. Chem. 277 (40) : 37169-75 (2002)]. Lysates were then subjected to polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) , followed by immunoblotting with one of the following antibodies (Abs) : 1:2,500 pan-Ras Ab; 1:50 anti-K-Ras Ab; 1:1000 anti- -tubulin Ab; 1:1000 anti-Gal-3 Ab; 1 1:10,000 anti-phospho-ERK Ab; 1:2,000 anti-ERK Ab; 1:750 anti-p21 Ab; 1:500 anti-Ttfl Ab. Immunoblots were then exposed to 1:5,000 peroxidase-goat anti-mouse IgG, 1:5,000 peroxidase- goat anti-rabbit IgG, or 1:5,000 peroxidase-goat anti-rat IgG, and protein bands were visualized using an enhanced chemiluminescence (ECL) kit (Amersham Pharmacia Biotech, Arlington Heights, IL) .

[0051] Ras-GTP Assays

[0052] Lysates containing 1 mg protein were used for determination of Ras-GTP by the glutathione S-transferase (GST)-RBD pull-down assay as previously described [Elad-Sfadia, et al. (2002), supra.], followed by Western immunoblotting with Ras isoform-specific Abs as described above .

[0053] Confocal microscopy.

[0054] 2 x 10⁵ ARO cells were plated on glass coverslips and then were treated with 75 μ FTS or the vehicle (0.1% DMSO) . The cells were fixed after 72 hours, and then permeabilized with 0.5% Triton X-100. Samples were blocked with 2% bovine serum albumin and 200 μg/ml goat gamma globulin for 30 min. The cells were labeled with 1 g/mL anti Ttf-1, Gal-3 and pan-Ras antibodies by for 1 hour and then with 1:750 goat anti rabbit fluorescein, goat anti-rat fluorescein and donkey anti- mouse cy3 Abs (Jackson ImmunoResearch) respectively. Each of the incubations was followed by three extensive washes. Staining intensity was analyzed with a Meta Zeiss LSM 510 confocal microscope. Quantification of the Ttf-1 in the nucleus of each cell was done by ImageJ software.

[0055] Infections and shRNAs

[0056] Viruses were produced by transient triple- transfections of HEK 293 cells using 6 μg retroviral vectors encoding for specific shRNA against Ttf-1 (clone ID V2HS_61850 Open-Biosystems ) with 3μg pMD2G and 3 μg pCGP encoding the retroviral envelope and the Gag and Pol proteins, respectively. As a control, we used 6 μg of no-silencing shRNA (Open-Biosystems) as previously described [Shalom-Feuerstein, et al., Biochim. Biophys. Acta, (2008)].

[0057 ] Results

[0058] Galectin-3 expression in thyroid carcinoma correlates with high levels of K-Ras.GTP and with growth inhibition by FTS.

[0059] In the first set of experiments we examined the effect of the Ras inhibitor FTS on growth of the 4 thyroid carcinoma cell lines namely the NPA (papillary) , MRO (follicular), TT (medullary) and ARO (anaplastic) lines. We examined the impact of the Ras inhibitor FTS on cell proliferation using the BrdU assay. FTS strongly inhibited in a dose-dependent manner incorporation of BrdU into DNA (Fig. 1A) and induced cell death (indicated by the cell viability reagent AlamarBlue; Fig. IB) in all cell lines except for TT cells. In the latter, FTS caused only weak inhibition of cell growth .

[0060] When we determined the levels of active Ras using the RBD pull down assay and pan Ras antibody, we found that ARO, MRO and NPA exhibited relatively high levels of Ras.GTP while TT exhibited relatively low levels of Ras.GTP (Fig. 2) . None of the cell lines under study has the Ras mutation [Namba, et al., J. Clin. Endocrinol. Metab. 88(9): 4393-7 (2003); [Nikiforova, et al., J. Clin. Endocrinol. Metab. 88 (11) :5399-404 (2003).] Therefore, the relatively high levels of Ras.GTP observed in these thyroid carcinomas are most likely due to stimulation of Ras exchange factors by growth factor receptors [Kolibaba, et al., Biochim. Biophys. Acta. 1333 (3) : F217-48 (1997); Huang, et al., J. Biol. Chem. 272(5) :2927-35 (1997); Smith, et al., Proc. Natl. Acad. Sci U S A 84 (21) : 7567-70 (1987)] and possibly due to Ras chaperons that stabilize the active Ras.GTP. [Elad-Sfadia, et al.,. J. Bol. Chem. 279 (33) : 34922-30 (2004); Elad-Sfadia, et al., J. Biol. Chem. 277 (40) : 37169-75 (2002).] [0061] Thus, we next examined next the levels of Gal-3 which is known to be involved in thyroid malignancies [Saggiorato, et al., J. Clin. Endocrinol. Metab. 86 (11) : 5152-8 (2001); Inohara, et al., Cancer 85 (11) : 2475-84 (1999); Orlandi, et al., Cancer Research 58 (14) : 3015-20 (1998); Saggiorato, et al., J. Endocrinol. Invest. 27 (4) :311-7 (2004)] and is a known chaperon of K-Ras.GTP [Elad-Sfadia, et al., J. Bol. Chem. 279 (33) : 34922-30 (2004); Shalom-Feuerstein, et al., Cancer Res. 68 (16) : 6608-16 (2008).] We found that ARO, MRO and NPA cells expressed relatively high levels of Gal-3 while TT cells did not express Gal-3 at all (Fig. 2). Interestingly, while we could not detect significant amount of H-Ras in any of the thyroid carcinoma cell lines (not shown) we detected N-Ras protein (Fig. 2). However, levels of N-Ras.GTP did not correlate with the level of Gal-3 (Fig. 2). Interestingly NPA cells exhibited the highest levels of p-ERK (Fig. 2) possibly due to two, factors, namely: i) the chronically active Ras that they possess; and ii) the activating B-Raf mutations they carry in two alleles [Liu, et al., Thyroid 18 (8) : 853-64 (2008); Carta, et al. , Clin. Endocrinol. (Oxf) 64 (1): 105-9 (2006).] We thus continued to study mainly the ARO (B- Raf mutation in one allele only, and MRO (no B-Raf mutation [Liu, et al., Thyroid 18(8): 853-64 (2008); Carta, et al., Clin. Endocrinol. (Oxf) 64 (1) : 105-9 (2006).].

[0062] FTS downregulates K-Ras.GTP and affects K-Ras signaling to ERK in ARO and MRO cells that exhibit high Gal-3.

[0063] We then examined the impact of FTS on the levels of Ras.GTP in the various thyroid carcinoma cell lines. Typical results of these experiments indicated that FTS (75 μΜ for 48 h) reduced the levels of Ras.GTP and had only a small effect on total Ras (Fig. 3A) . A separate experiment showed that FTS reduced the levels of K-Ras.GTP (Fig. 3B) . This later experiment was performed with serum-starved cells in order to allow determination of the impact of FTS under basal conditions, namely without the stimulatory effects of the serum. As shown, the levels of K-Ras.GTP were relatively high in ARO, MRO and NPA even under serum starvation and FTS reduced K-Ras.GTP (Fig. 3B) .

[0064] Next we examined whether the reduced K-Ras.GTP levels in thyroid carcinoma cells treated with FTS is translated into reduced Ras signaling. We therefore examined the levels of phospho-ERK and phospho-Akt as readouts of the two prominent Ras pathways Raf-MEK-ERK and PI3-K-Akt respectively. Phospho-Akt was not detected in any of the cell lines even without drug treatment (not shown) . Therefore phosphor-Akt could not be used as readout for Ras signaling. Nonetheless, as described above (Fig. 2) , we detected phospho- ERK in all cell lines, and observed that FTS caused a strong significant reduction in the levels of phospho-ERK in ARO and MRO cells Fig. 3C) . FTS effected no reduction of phospho-ERK in NPA and TT cells (Fig. 3C) . The relatively small effect of FTS on phospho-ERK in NPA cells whose high K-Ras.GTP was downregulated by FTS is most likely attributed to the belief that these cells carry activating B-Raf mutations (V600E) in both alleles [Carta, et al., Clin. Endocrinol. (Oxf) 64(1): 105-9 (2006)]. Therefore in NPA cells there is a relatively strong Raf signal to ERK which is independent of active Ras. This is not the case of ARO cells that are heterozygous with respect to activated mutant B-Raf and carry one wt B-Raf allele and MRO cells that carry no B-Raf mutations [Liu, et al., Thyroid 18 (8) : 853-64 (2008; Carta, et al., (2006)). Thus, in both cell lines, the Ras-dependent activation of the wild-type (wt) B-Raf is inhibited when the levels of K-Ras.GTP are downregulated by FTS.

[0065] FTS upregulates the cell cycle inhibitor p21 and the thyroid transcription factor l(Ttf-l).

[0066] Earlier studies with cancer cell lines with active Ras showed that the cell cycle inhibitor p21 which inhibits CDK2 is, at least in part, negatively regulated by active Ras [Halaschek-Wiener, et al., Cell Signal 16 (11) : 1319-27 (2004)], and that FTS increased the levels of p21 in a number of cancer cell lines [Halaschek, et al., Mol . Med. 6 (8) : 693-704 (2000)]. We therefore examined whether FTS affects p21 levels in the thyroid cell lines under study. We found that FTS increased the levels of p21 in ARO, MRO and NPA but not in TT cells (Fig. 4A) which positively correlated with reduction in the levels of K-Ras.GTP and inhibition of cell growth in ARO, MRO and NPA only (Figs. 1 and 3A) . It thus appears that the FTS induced increase in p21 is a major factor of cell growth inhibition in thyroid cells with high Gal-3.

[0067] Next we examined whether FTS affects the levels of Ttf-1 which is negatively regulated in thyroid carcinomas by the Raf-MEK-ERK pathway [Missero, et al., Molecular and Cellular Biology 20 (8) : 2783-93 (2000)]. We found that FTS upregulated Ttf-1 in ARO and MRO cells but not in NPA and TT cells (Fig. 4A' ) . This is again consistent with our results that K-Ras.GTP and Ras-dependent ERK activation are more sensitive to FTS in ARO and MRO than in the other cell lines. Like FTS, dominant-negative (DN) -Ras upregulated Ttf-1 in ARO and MRO cells (Fig. 4B) .

[0068] Our results thus suggested that Ras inhibition by FTS or by DN-Ras reverts, at least in part, the malignant phenotype of the most malignant cell line studied here (ARO) by arresting cell growth and increasing the differentiation transcription factor Ttf-1 known as a critical factor in thyroid cells [Ros, et al., Biochimie 81 (4) : 389-96 (1999); Missero, et al., Molecular and Cellular biology 20 (8) : 2783-93 (2000); DeVita, et al., Molecular Endocrinology 19(1) :76-89 (2005); Akagi, et al., British Journal of Cancer 99 (5): 781-8 (2008)]. Indeed when we examined FTS-treated ARO cells microscopically, we found a change in their morphology where the cells became more spread and less clustered (not shown) . In addition we observed a marked increase in nuclear Ttf-1 in these cells after FTS treatment and some, yet unexplained, increase in paranuclear Ttf-1 (Fig. 4C) . Like FTS, the MEK inhibitor U0126 caused a marked increase in the levels of p21 and Ttf-1 in ARO and MRO cells (Figs. 4D and D') suggesting that these increases are mediated through the inhibition of the Ras-Raf-MEK-ERK pathway. These results support the notion that mechanism of FTS action involves at least in part upregulation of Ttf-1.

[0069] Altogether the results described above suggested that Gal-3 interactions with K-Ras.GTP in ARO and MRO cells result in a robust signal to the Raf-MEK-ERK cascade which is known to negatively regulate p21 and Ttf-1, hence to induce rapid inhibition cell growth and induction of differentiation.

[0070] FTS disrupts K-Ras-Gal-3 co-localization in the cell membrane of ARO cells.

[0071] We next examined whether FTS disrupts the interaction of K-Ras and Gal-3 in the cell membrane. To this end, we used the most malignant cells in our series, the ARO cells and stained them with mouse anti-pan Ras antibody and with rat anti-Gal-3 antibody before and after FTS treatment. The cells were then stained with cy3-labeled anti-mouse antibodies (Ras labeling) and with fluorescein labeled anti- rat antibody (Gal-3 labeling) (photos not shown) . Typical fluorescence confocal images of these experiments showed that Ras was localized mainly to the cell membrane of the control cells (not shown) and that after FTS treatment a major fraction of Ras was misslocalized to the cytoplasm (not shown) . Importantly, Gal-3 in ARO cells was found to be localized both to the cytoplasm and to the cell membrane (not shown) and after FTS treatment most of the Gal-3 was cytoplasmic (not shown) . The strong impact of FTS on endogenous Ras and Gal-3 interactions is clearly demonstrated by the observed disruption of Gal-3 and Ras colocalization in the plasma membrane of the drug treated cells (not shown) . Statisitical analysis of the results is shown in Fig. 5. Importantly, these results demonstrate disruption of the interaction between Ras and Gal-3 in cancer cells without exogenous expression of the two binding partners.

[0072] Example 2 — Animal experiments

[0073] Next we examined whether FTS can inhibit thyroid carcinoma growth in vivo. To this end, we used the most malignant cell line in our arsenal, the ARO cells, and implanted the cells under the skin in the flank of male nude mice as detailed previously [Barkan, et al . , Clin. Cancer Res. 12 (18) : 5533-42 (2006)]. Seven days after cell implantation, the volumes of the tumors were 0.5-0.6 cm³. The mice were then divided randomly into the control (10 vehicle-treated mice) and FTS-treated groups (10 mice). The mice received 25 days orally FTS (60 mg/kg, daily) or the vehicle and tumor volumes were determined at the indicated times. The mice were then sacrificed for the determination of tumor weight and pharmacodynamics. Results are shown in Figs. 6A-C. As shown, FTS caused a significant reduction in the rate of tumor growth (p< 0.05, Fig. 6A) . Tumor weight at the end point was also significantly smaller in the FTS-treated group as compared with the control (p= 0.01, Fig. 6B) . Fig. 6C shows the results of the pharmacodynamics. We found that the oral FTS treatment caused a significant reduction in the levels of Ras.GTP, Gal-3 and p-ERK. All together, these experiments showed that FTS hits its target in vivo in the tumors and inhibited growth of the anaplastic thyroid tumor.

[0074] All patent publications and non-patent publications are indicative of the level of skill of those skilled in the art to which this invention pertains. All these publications are herein incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference. [0075] Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims.

Claims

1. A method for treating thyroid cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a Ras antagonist represented by the formula

wherein represents wherein R¹ represents farnesyl, or geranyl- geranyl; R² is COOR⁷, CONR⁷R⁸, or COOCHR⁹OR¹⁰, wherein R⁷ and R⁸ are each independently hydrogen, alkyl, or alkenyl; wherein R⁹ represents H or alkyl; and wherein R¹⁰ represents alkyl; and wherein R³, R⁴, R⁵ and R⁶ are each independently hydrogen, alkyl, alkenyl, alkoxy, halo, trifluoromethyl , trifluoromethoxy, or alkylmercapto; and wherein X represents S; and wherein the thyroid cancer is characterized by elevated levels of K-Ras.

2. The method of claim 1, wherein the Ras antagonist is FTS.

3. The method of claim 1, wherein the Ras antagonist is an FTS analog selected from the group consisting of 5-chloro-FTS, 5-fluoro-FTS, FTS-methyl ester, FTS-amide, FTS-methylamide, FTS-dimethylamide, methoxymethyl S-farnesylthiosalicylate, methoxymethyl S-geranylgeranylthiosalicylate, methoxymethyl 5- fluoro-S-farnesylthiosalicylate, and ethoxymethyl S- farnesylthiosalicyate .

4. The method of any one of claims 1-3, wherein the administering is oral.

5. The method of claim 4, wherein the Ras antagonist is administered in an oral dosage form which is a tablet or capsule .

6. The method of any of claims 1-5, wherein the thyroid cancer is papillary thyroid carcinoma.

7. The method of any of claims 1-5, wherein the thyroid cancer is follicular thyroid carcinoma.

8. The method of any of claims 1-5, wherein the thyroid cancer is anaplastic thyroid carcinoma.

9. A Ras antagonist in an effective amount represented by the formula

wherein represents wherein R¹ represents farnesyl, or geranyl- geranyl; R² is COOR⁷, CONR⁷R⁸, or COOCHR⁹OR¹⁰, wherein R⁷ and R⁸ are each independently hydrogen, alkyl, or alkenyl; wherein R⁹ represents H or alkyl; and wherein R¹⁰ represents alkyl; and wherein R³, R⁴, R⁵ and R⁶ are each independently hydrogen, alkyl, alkenyl, alkoxy, halo, trifluoromethyl , trifluoromethoxy, or alkylmercapto; and wherein X represents S; for use in the treatment of thyroid cancer in a patient in need thereof; and

wherein the thyroid cancer is characterized by elevated levels of K-Ras.

10. The Ras antagonist of claim 9, wherein the Ras antagonist is FTS.

11. The Ras antagonist of claim 9, wherein the Ras antagonist is an FTS analog selected from the group consisting of 5- chloro-FTS, 5-fluoro-FTS, FTS-methyl ester, FTS-amide, FTS- methylamide, FTS-dimethylamide, methoxymethyl S- farnesylthiosalicylate, methoxymethyl S- geranylgeranylthiosalicylate, methoxymethyl 5-fluoro-S- farnesylthiosalicylate, and ethoxymethyl S- farnesylthiosalicyate .

12. The Ras antagonist of any one of claims 9-11, wherein the administering is oral.

13. The Ras antagonist of claim 4, wherein the Ras antagonist is administered in an oral dosage form which is a tablet or capsule .

14. The Ras antagonist of any of claims 9-13, wherein the thyroid cancer is papillary thyroid carcinoma.

15. The Ras antagonist of any of claims 9-13, wherein the thyroid cancer is follicular thyroid carcinoma.

16. The Ras antagonist of any of claims 9-13, wherein the thyroid cancer is anaplastic thyroid carcinoma.

17. A pharmaceutical composition for treating thyroid cancer in a patient in need thereof, the composition comprising a Ras antagonist represented by the formula

wherein represents wherein R¹ represents farnesyl, or geranyl- geranyl; R² is COOR⁷, CONR⁷R⁸, or COOCHR⁹OR¹⁰, wherein R⁷ and R⁸ are each independently hydrogen, alkyl, or alkenyl; wherein R⁹ represents H or alkyl; and wherein R¹⁰ represents alkyl; and wherein R³, R⁴, R⁵ and R⁶ are each independently hydrogen, alkyl, alkenyl, alkoxy, halo, trifluoromethyl, trifluoromethoxy, or alkylmercapto; and wherein X represents S; and wherein the thyroid cancer is characterized by elevated levels of K-Ras.

18. The pharmaceutical composition of claim 17, wherein the Ras antagonist is FTS.

19. The pharmaceutical composition of claim 17, wherein the Ras antagonist is an FTS analog selected from the group consisting of 5-chloro-FTS, 5-fluoro-FTS, FTS-methyl ester, FTS-amide, FTS-methylamide, FTS-dimethylamide, methoxymethyl S-farnesylthiosalicylate, methoxymethyl S- geranylgeranylthiosalicylate, methoxymethyl 5-fluoro-S- farnesylthiosalicylate, and ethoxymethyl S- farnesylthiosalicyate .

20. The pharmaceutical composition of any one of claims 17- 19, wherein the composition is suitable for oral administration.

21. The pharmaceutical composition of claim 20, wherein the Ras antagonist is in the form of an oral dosage form which is a tablet or capsule.

22. The pharmaceutical composition of any of claims 17-21, wherein the thyroid cancer is papillary thyroid carcinoma.

23. The pharmaceutical composition of any of claims 17-21, wherein the thyroid cancer is follicular thyroid carcinoma.

24. The pharmaceutical composition of any of claims 17-21, wherein the thyroid cancer is anaplastic thyroid carcinoma.