Synchronous renal malignancy presenting as recurrent urinary tract infections.

Renal cell carcinoma (RCC) and urothelial carcinoma of the upper urinary tract are not uncommon urological malignancies. Their simultaneous occurrence in a patient is, however, extraordinarily rare. We report the case of a patient who underwent unilateral nephrectomy for suspected RCC and diagnosed transitional cell carcinoma of the superior pelvis. Preoperative imaging was suspicious for renal pelvic involvement, which was confirmed upon performing cystoscopy and biopsy of the suspected lesion preoperatively. This preoperative approach was especially appropriate as a nephron saving procedure was being considered prior to the discovery of the synchronous lesion. We discuss this rare simultaneous occurrence of synchronous malignancies in the same kidney.

Evaluation of farnesyl:protein transferase and geranylgeranyl:protein transferase inhibitor combinations in preclinical models.

Farnesyl:protein transferase (FPTase) inhibitors (FTIs) were originally developed as potential anticancer agents targeting the ras oncogene and are currently in clinical trials. Whereas FTIs inhibit the farnesylation of Ha-Ras, they do not completely inhibit the prenylation of Ki-Ras, the allele most frequently mutated in human cancers. Whereas farnesylation of Ki-Ras is blocked by FTIs, Ki-Ras remains prenylated in FTI-treated cells because of its modification by the related prenyltransferase, geranylgeranyl:protein transferase type I (GGPTase-I). Hence, cells transformed with Ki-ras tend to be more resistant to FTIs than Ha-ras-transformed cells. To determine whether Ki-ras-transformed cells can be targeted by combining an FTI with a GGPTase-I inhibitor (GGTI), we evaluated potent, selective FTIs, GGTIs, and dual prenylation inhibitors (DPIs) that have both FTI and GGTI activity. We find that in human PSN-1 pancreatic tumor cells, which harbor oncogenic Ki-ras, and in other tumor lines having either wild-type or oncogenic Ki-ras, treatment with an FTI/GGTI combination or with a DPI blocks Ki-Ras prenylation and induces markedly higher levels of apoptosis relative to FTI or GGTI alone. We demonstrate that these compounds can inhibit their enzyme targets in mice by monitoring pancreatic and tumor tissues from treated animals for inhibition of prenylation of Ki-Ras, HDJ2, a substrate specific for FPTase, and Rap1A, a substrate specific for GGPTase-I. Continuous infusion (72 h) of varying doses of GGTI in conjunction with a high, fixed dose of FTI causes a dose-dependent inhibition of Ki-Ras prenylation. However, a 72-h infusion of a GGTI, at a dose sufficient to inhibit Ki-Ras prenylation in the presence of an FTI, causes death within 2 weeks of the infusion when administered either as monotherapy or in combination with an FTI. DPIs are also lethal after a 72-h infusion at doses that inhibit Ki-Ras prenylation. Because 24 h infusion of a high dose of DPI is tolerated and inhibits Ki-Ras prenylation, we compared the antitumor efficacy from a 24-h FTI infusion to that of a DPI in a nude mouse/PSN-1 tumor cell xenograft model and in Ki-ras transgenic mice with mammary tumors. The FTI and DPI were dosed at a level that provided comparable inhibition of FPTase. The FTI and the DPI displayed comparable efficacy, causing a decrease in growth rate of the PSN-1 xenograft tumors and tumor regression in the transgenic model, but neither treatment regimen induced a statistically significant increase in tumor cell apoptosis. Although FTI/GGTI combinations elicit a greater apoptotic response than either agent alone in vitro, the toxicity associated with GGTI treatment in vivo limits the duration of treatment and, thus, may limit the therapeutic benefit that might be gained by inhibiting oncogenic Ki-Ras through dual prenyltransferase inhibitor therapy.

The synthesis of a prodrug of doxorubicin designed to provide reduced systemic toxicity and greater target efficacy.

Doxorubicin (Dox) can provide some stabilization in prostate cancer; however, its use is limited because of systemic toxicities, primarily cardiotoxicity and immunosuppression. The administration of a prodrug of doxorubicin, designed to permit selective activation by the tumor, would reduce general systemic exposure to the active drug and would thereby increase the therapeutic index. Prostate specific antigen (PSA) is a serine protease with chymotrypsin-like activity that is a member of the kallikrein gene family. PSA's putative physiological role is the liquefaction of semen by virtue of its ability to cleave the seminal fluid proteins semenogelins I and II. Serum PSA levels have been found to correlate well with the number of malignant prostate cells. The use of a prodrug which is cleaved by the enzyme PSA in the prostate should in principle produce high localized concentrations of the cytotoxic agent at the tumor site while limiting systemic exposure to the active drug. Cleavage maps following PSA treatment of human semenogelin were constructed. Systematic modification of the amino acid residues flanking the primary cleavage site led to the synthesis of a series of short peptides which were efficiently hydrolyzed by PSA. Subsequent coupling of selected peptides to doxorubicin provided a series of doxorubicin-peptide conjugates which were evaluated in vitro and in vivo as targeted prodrugs for PSA-secreting tumor cells. From these studies we selected Glutaryl-Hyp-Ala-Ser-Chg-Gln-Ser-Leu-Dox, 27, as the peptide-doxorubicin conjugate with the best profile of physical and biological properties. Compound 27 has a greater than 20-fold selectivity against human prostate PSA-secreting LNCaP cells relative to the non-PSA-secreting DuPRO cell line. In nude mouse xenograft studies, 27 reduced PSA levels by 95% and tumor weight by 87% at a dose below its MTD. Both doxorubicin and Leu-Dox (13) were ineffective in reducing circulating PSA and tumor burden at their maximum tolerated doses. On the basis of these results, we selected 27 for further study to assess its ability to inhibit human prostate cancer cell growth and tumorigenesis.

Signaling pathways in apoptosis as potential targets for cancer therapy.

Genetic instability contributes to the origin of cancer as well as to the ability of cancer cells to become resistant to various therapies. Because of this, cytotoxic rather than cytostatic therapies might be most effective against this disease. Many oncogenes and tumor suppressors mediate their effects by interfering with or inducing apoptotic signaling. Thus, apoptotic pathways might be significantly altered in cancer cells relative to untransformed cells, and these differences might present a therapeutic window that can be exploited for development of cancer drugs.

PSA-specific and non-PSA-specific conversion of a PSA-targeted peptide conjugate of doxorubicin to its active metabolites.

Tumor-selective delivery of doxorubicin by a prostate-specific antigen (PSA)-targeted peptide conjugate prodrug of doxorubicin was demonstrated in a nude mouse xenograft model of human prostate cancer. The prodrug (referred to as doxorubicin conjugate) contains doxorubicin linked to a seven-amino acid peptide conjugate that was designed to increase delivery of doxorubicin to tumor sites through the hydrolytic properties of PSA, which prostate tumors express in high amounts. Following i.p. administration of the doxorubicin conjugate to mice, tumor exposure to doxorubicin was increased 2.5-fold as compared with that achieved after an equimolar dose of doxorubicin itself. However, in heart tissue, the site of clinical dose-limiting toxicity, doxorubicin concentrations observed after administration of doxorubicin conjugate were substantially lower than those in mice that received doxorubicin itself. While the prodrug provided selective delivery of doxorubicin to tumor tissue, there was substantial non-PSA-specific formation of doxorubicin in laboratory animals, a factor that would limit the extent of therapeutic gain of the prodrug. Following i.v. administration to mice, rats, dogs, and monkeys, about one-third of the dose was metabolized to doxorubicin. In tumor-bearing mice, the fraction of the dose metabolized to doxorubicin appeared even higher. This is likely the result of conjugate conversion to doxorubicin by both PSA-specific (in tumor) and non-PSA-specific proteolytic activities. In vitro studies provided further support for the PSA specificity of metabolism; LNCaP cells mediated rapid metabolism of the conjugate, while DuPRO-1 cells, which are deficient in PSA, were incapable of metabolism.

Drug-targeting strategies in cancer therapy.

Genetic changes in cell-cycle, apoptotic, and survival pathways cause tumorigenesis, leading to significant phenotypic changes in transformed cells. These changes in the tumor environment - elevated expression of surface proteases, increased angiogenesis and glucuronidase activity - can be taken advantage of to improve the therapeutic index of existing cancer therapies. Targeting cytotoxics to tumor cells by enzymatic activation is a promising strategy for improving chemotherapeutics.

Farnesyl:protein transferase inhibitors as potential agents for the management of human prostate cancer.

The effects of farnesyl:protein transferase inhibitors (FTIs) were evaluated against hormone-dependent and hormone-independent prostate cancer cell lines harboring mutant and wild type Ras. The combinations of the FTI with hormones and chemotherapy were explored. The effect of FTI on the growth of human prostate cancer lines was examined under anchorage-dependent and -independent conditions. Changes in Ras processing and cellular localization were examined by immunoblotting and immunocytochemistry. Hormone-dependent (LNCaP) and -independent (TSU-Pr1, PC3 and DU145) human prostate cancer cell lines were growth-inhibited by the FTI L-744,832 at concentrations ranging from 100 nM to 20 &mgr;M. The inhibition was accompanied by loss of protein farnesylation and with the accumulation of Ha-Ras as its unprocessed, cytosolic form. No effect on N- and Ki-Ras processing was observed. The transformed phenotype of TSU-Pr1 cells, which possess a Ha-Ras Gly-12-Val activating mutation, reverted following FTI treatment. Enhanced antitumor effects were observed when the FTI was combined with gamma-radiation, etoposide, doxorubicin, cisplatin, estramustine and the antihormone bicalutamide. In particular, the combination of taxol and FTI was synergistic for DU145 cells, a cell line that is only marginally sensitive to the FTI alone. The sensitivity of human prostate cancer cell lines to the FTI is independent of the presence of mutations of tumor suppressors, cell cycle regulators and of the activation of a variety of oncogenes, including Ras. A cell line expressing mutated Ha-Ras is particularly sensitive. Enhanced antitumor effects were observed with an anti-androgen, gamma-irradiation, and several chemotherapeutic agents. These findings support the clinical evaluation of FTIs alone or in combination as treatment for this disease. Prostate Cancer and Prostatic Diseases (2001) 4, 33-43

A peptide-doxorubicin 'prodrug' activated by prostate-specific antigen selectively kills prostate tumor cells positive for prostate-specific antigen in vivo.

We covalently linked doxorubicin with a peptide that is hydrolyzable by prostate-specific antigen. In the presence of prostate tumor cells secreting prostate-specific antigen, the peptide moiety of this conjugate, L-377,202, was hydrolyzed, resulting in the release of leucine-doxorubicin and doxorubicin, which are both very cytotoxic to cancer cells. However, L-377,202 was much less cytotoxic than conventional doxorubicin to cells in culture that do not secrete prostate-specific antigen. L-377,202 was approximately 15 times more effective than was conventional doxorubicin at inhibiting the growth of human prostate cancer tumors in nude mice when both drugs were used at their maximally tolerated doses. Nude mice inoculated with human prostate tumor cells secreting prostate-specific antigen showed considerable reductions in tumor burden with minimal total body weight loss when treated with L-377, 202. This improvement in therapeutic index correlated with the selective localization of leucine-doxorubicin and free doxorubicin in tissues secreting prostate-specific antigen after exposure to L-377,202.

Farnesyltransferase inhibitors potentiate the antitumor effect of radiation on a human tumor xenograft expressing activated HRAS.

Successful radiosensitization requires that tumor cells become more radiosensitive without causing an equivalent reduction in the survival of cells of the surrounding normal tissues. Since tumor cell radiosensitivity can be influenced by RAS oncogene activation, we have hypothesized that inhibition of oncogenic RAS activity would lead to radiosensitization of tumors with activated RAS. We previously showed in tissue culture that prenyltransferase treatment of cells with activated RAS resulted in radiosensitization, whereas treatment of cells with wild-type RAS had no effect on radiation survival. Here we ask whether the findings obtained in vitro have applicability in vivo. We found that treatment of nude mice bearing T24 tumor cell xenografts with farnesyltransferase inhibitors resulted in a significant and synergistic reduction in tumor cell survival after irradiation. The regrowth of T24 tumors expressing activated RAS was also significantly prolonged by the addition of treatment with farnesyltransferase inhibitors compared to the regrowth after irradiation alone. In contrast, there was no effect on the radiosensitivity of HT-29 tumors expressing wild-type RAS. These results demonstrate that specific radiosensitization of tumors expressing activated RAS oncogenes can be obtained in vivo.

Mouse mammary tumor virus-Ki-rasB transgenic mice develop mammary carcinomas that can be growth-inhibited by a farnesyl:protein transferase inhibitor.

For Ras oncoproteins to transform mammalian cells, they must be posttranslationally modified with a farnesyl group in a reaction catalyzed by the enzyme farnesyl:protein transferase (FPTase). Inhibitors of FPTase have therefore been developed as potential anticancer agents. These compounds reverse many of the malignant phenotypes of Ras-transformed cells in culture and inhibit the growth of tumor xenografts in nude mice. Furthermore, the FPTase inhibitor (FTI) L-744,832 causes tumor regression in mouse mammary tumor virus (MMTV)-v-Ha-ras transgenic mice and tumor stasis in MMTV-N-ras mice. Although these data support the further development of FTIs, it should be noted that Ki-ras is the ras gene most frequently mutated in human cancers. Moreover, Ki-RasB binds more tightly to FPTase than either Ha- or N-Ras, and thus higher concentrations of FTIs that are competitive with the protein substrate may be required to inhibit Ki-Ras processing. Given the unique biochemical and biological features of Ki-RasB, it is important to evaluate the efficacy of FTIs or any other modulator of oncogenic Ras function in model systems expressing this Ras oncoprotein. We have developed strains of transgenic mice carrying the human Ki-rasB cDNA with an activating mutation (G12V) under the control of the MMTV enhancer/promoter. The predominant pathological feature that develops in these mice is the stochastic appearance of mammary adenocarcinomas. High levels of the Ki-rasB transgene RNA are detected in these tumors. Treatment of MMTV-Ki-rasB mice with L-744,832 caused inhibition of tumor growth in the absence of systemic toxicity. Although FPTase activity was inhibited in tumors from the treated mice, unprocessed Ki-RasB was not detected. These results demonstrate the utility of the MMTV-Ki-rasB transgenic mice for testing potential anticancer agents. Additionally, the data suggest that although the FTI L-744,832 can inhibit tumor growth in this model, Ki-Ras may not be the sole mediator of the biological effects of the FTI.

Farnesyltransferase inhibitors: antineoplastic properties, mechanisms of action, and clinical prospects.

Farnesyltransferase (FTase) inhibitors are among the current wave of molecularly targeted anti-cancer agents being used to attack malignancy in a rational manner. A large body of preclinical data indicates that FTase inhibitors block cancer cell proliferation through both cytostatic and cytotoxic effects. Interestingly, FTase inhibitors have rather limited effects on normal cell function, suggesting that they may target unique aspects of cancer cell pathophysiology. The development of FTase inhibitors was predicated on the discovery that the Ras oncoproteins must be post-translationally modified to transform cells. However, recent work indicates that the anti-neoplastic effects of FTase inhibitors depend on altering the post-translational modifications of non-Ras proteins as well. In particular, a critical target protein that responds to FTase inhibition by blocking tumor cell growth is RhoB, an endosomal Rho protein that functions in receptor trafficking. In this review, we survey the biological foundations for the clinical development of FTase inhibitors, and consider some of the latest mechanistic studies that reveal how these agents affect cellular physiology.

Imidazole-containing diarylether and diarylsulfone inhibitors of farnesyl-protein transferase.

The design and syntheses of non-thiol inhibitors of farnesyl-protein transferase are described. Optimization of cysteine-substituted diarylethers led to highly potent imidazole-containing diarylethers and diarylsulfones. Polar diaryl linkers dramatically improved potency and gave highly cell active compounds.

Design and in vivo analysis of potent non-thiol inhibitors of farnesyl protein transferase.

Inhibitors of farnesyl protein transferase (FPTase) based upon a pseudotripeptide template are described that comprise an imidazole group substituted with a hydrophobic substituent. (1, 5)-Disubstitution of the imidazole group is shown to be the optimal array that leads to potent and selective inhibitors of FPTase. A variety of aryl and isoprenyl substituents are shown to afford effective inhibitors, and the mechanism by which these compounds inhibit FPTase has been investigated. The biochemical behavior of these compounds suggests that they bind to FPTase at the site usually occupied by the protein substrate. In experiments in cell culture, the methyl ester prodrugs of these inhibitors are cell permeant and potently inhibit the posttranslational modification of H-Ras protein. Additionally, these molecules revert the phenotype of ras transformed cells as evidenced by their ability to slow the growth of ras transformed cell lines in soft agar. One of the inhibitors, as its methyl prodrug, was evaluated in two in vivo models of tumor growth. The compound selectively inhibited the growth of tumors derived from H-ras transformed cells, in nude mice, and caused the regression of preexisting tumors in an H-ras transgenic animal model.

Non-thiol 3-aminomethylbenzamide inhibitors of farnesyl-protein transferase.

The design and syntheses of non-thiol inhibitors of farnesyl-protein transferase are described. Substitutions on an imidazolylmethyl-AMBA-methionine template gave a highly potent and cell-active inhibitor.

Treatment with farnesyl-protein transferase inhibitor induces regression of mammary tumors in transforming growth factor (TGF) alpha and TGF alpha/neu transgenic mice by inhibition of mitogenic activity and induction of apoptosis.

Mouse mammary tumor virus-transforming growth factor alpha (MMTV-TGF alpha) and MMTV-TGF alpha/neu transgenic mice develop mammary tumors after a long latency and therefore provide useful model systems for breast cancer with its recognized activation of receptor tyrosine kinase signaling. We used these mice to study the antitumor effect of L-744,832 (FTI), a potent and selective inhibitor of farnesyl-protein transferase, and hence of Ras function. A total of 55 mice were assigned randomly to treatment with FTI or vehicle, and one-half of the mice were crossed over after initial treatment to the opposite group. L-744,832 induced reversible regression of mammary tumors that was paralleled by a decrease in serum levels of TGF alpha secreted by the tumor cells. There was no difference in response to treatment with FTI between MMTV-TGF alpha mice, in which tumorigenesis was accelerated by multiparity or the chemical carcinogen 7,12-dimethylbenzanthracene, and MMTV-TGF alpha/neu mice. The tumor histological type had no impact on FTI sensitivity. For mechanistic analyses, tumor excision biopsies were obtained from 12 mice before and after treatment with L-744,832. In these samples, tumor regression was paralleled biochemically by inhibition of mitogen-activated protein kinase activity and biologically by an increase in G1-phase and decrease in S-phase fractions, as well as induction of apoptosis. These results suggest that the potential clinical use of FTI could be expanded to include cancers harboring activated receptor tyrosine kinases as well as those containing activated Ras.

N-Arylalkyl pseudopeptide inhibitors of farnesyltransferase.

Inhibitors of Ras protein farnesyltransferase are described which are reduced pseudopeptides related to the C-terminal tetrapeptide of the Ras protein that signals farnesylation. Reduction of the carbonyl groups linking the first three residues of the tetrapeptide leads to active inhibitors which are chemically unstable. Stability can be restored by alkylating the central amine of the tetrapeptide. Studies of the SAR of these alkylated pseudopeptides with concomitant modification of the side chain of the third residue led to 2(S)-(2(S)-¿[2(S)-(2(R)-amino-3-mercaptopropylamino)-3(S)- methylpentyl]naphthalen-1-ylmethylamino¿acetylamino)-4 -methylsulfany lbutyric acid (11), a subnanomolar inhibitor. The methyl ester (10) of this compound exhibited submicromolar activity in the processing assay and selectively inhibited anchorage-independent growth of Rat1 cells transformed by v-ras at 2.5-5 microM.

Biochemical and biological analyses of farnesyl-protein transferase inhibitors.

The methods outlined in Subheading 3. provide a logical sequence of assays with which to evaluate the biochemical and biological properties of potential FPTase inhibitors. The clinical predictability of these assays must await the evaluation of one or more of these compounds in humans.

Therapeutic intervention and signaling.

Significant advances in our understanding of intracellular signal transduction pathways have emerged within the past several years. It is now apparent that, under certain circumstances, particular isoforms of Ras can be prenylated by geranylgeranyl protein transferase as well as farnesyl protein transferase. New pathways controlling growth factor-dependent inhibition of apoptosis involving phosphoinositide 3'-hydroxykinase and the protein kinase Akt have also been clarified.

Farnesyl transferase inhibitors cause enhanced mitotic sensitivity to taxol and epothilones.

An important class of cellular proteins, which includes members of the p21ras family, undergoes posttranslational farnesylation, a modification required for their partition to membranes. Specific farnesyl transferase inhibitors (FTIs) have been developed that selectively inhibit the processing of these proteins. FTIs have been shown to be potent inhibitors of tumor cell growth in cell culture and in murine models and at doses that cause little toxicity to the animal. These data suggest that these drugs might be useful therapeutic agents. We now report that, when FTI is combined with some cytotoxic antineoplastic drugs, the effects on tumor cells are additive. No interference is noted. Furthermore, FTI and agents that prevent microtubule depolymerization, such as taxol or epothilones, act synergistically to inhibit cell growth. FTI causes increased sensitivity to induction of metaphase block by these agents, suggesting that a farnesylated protein may regulate the mitotic check point. The findings imply that FTI may be a useful agent for the treatment of tumors with wild-type ras that are sensitive to taxanes.