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The Oncologist, Vol. 1, No. 4, 255–259, August 1996
© 1996 AlphaMed Press

ADVANCES IN CANCER TREATMENT: THE CHABNER SYMPOSIUM

New Concepts for the Development and Use of Antifolates

Edward Chu, Jean L. Grem, Patrick G. Johnston, Carmen J. Allegra

NCI-Navy Medical Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA

Correspondence: Carmen J. Allegra, M.D., NCI-Navy Medical Oncology Branch, National Cancer Institute, National Institutes of Health, 8901 Wisconsin Avenue, Building 8, Room 5101, Bethesda, MD 20889-5105, USA. Telephone: 301-496-0901; Fax: 301-496-0047.


   ABSTRACT
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Approximately one-third of all cases of colorectal carcinoma present in an advanced and, therefore, incurable stage. For these patients, the development of new chemotherapeutic strategies is of central importance. Biochemical modulation of 5-fluorouracil (5-FU) has resulted in approximately a twofold increase in activity of 5-FU. Recent preclinical investigations suggest that interferon can also modulate the activity of 5-FU and may result in enhanced response rates in patients. One of the critical mechanisms of resistance to 5-FU appears to be the acute induction in thymidylate synthase (TS) levels following therapy with inhibitors of this enzyme. This mechanism is based on a novel autoregulatory feedback pathway wherein the TS protein regulates its own translational efficiency. Regulatory function of the enzyme is dependent on its state of occupancy by either the physiologic ligands or inhibitors, including fluoropyrimidines and antifolates. Ongoing efforts are directed toward utilizing knowledge of this protein/messenger RNA interaction for therapeutic benefit. Given the importance of TS, our laboratory has developed antibodies capable of quantitating the levels of this enzyme in fresh or paraffin-embedded tissues. Preliminary investigations suggest that the level of TS has prognostic importance in patients with rectal carcinoma and may be used to predict responsiveness to fluoropyrimidine agents. Novel strategies utilizing dual modulation of 5-FU with leucovorin and interferon are under investigation in both the advanced and adjuvant disease settings. Emerging mechanistic concepts regarding TS, along with the development of new, more potent inhibitors will hopefully result in future therapeutic gains.

Key Words. Antifolates • Thymidylate synthase • Enzyme regulation • Monoclonal antibodies • Immunohistochemistry • Interferon • Biochemical modulation

For patients with advanced colorectal carcinoma, 5-fluorouracil (5-FU) represents the most active single agent presently available. However, biochemical modulation of 5-FU with the reduced folate leucovorin (LV) (5-formyltetrahydrofolate) has significantly improved overall response rates to the 25%-30% range [1]. Unfortunately, therapy with this modality is not curative, and there is continued debate as to its ability to prolong life in patients with advanced disease. Despite 40 years of research dedicated to the development of new agents for the treatment of patients with colorectal carcinoma, only recently have agents been identified with promising clinical activity, including the camptothecin analog Irinotecan (CPT-11) and the antifolate analog Tomudex (ZD1694) [2, 3]. In addition to searching for novel compounds, significant research efforts have also focused on improving the antineoplastic activity of 5-FU through biochemical modulation.

Thymidylate synthase (TS) is a folate-dependent enzyme responsible for the de novo synthesis of thymidylate, a required nucleotide precursor for DNA replication and repair. This enzymatic reaction utilizes 2'-deoxyuridine-5'-monophosphate (dUMP) and 5,10-methylenetetrahydrofolate which serves as the one-carbon donor for the reductive methylation of dUMP. The 5-FU nucleotide metabolite 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) has been shown to form a covalent ternary complex with TS and the reduced folate 5,10-methylenetetrahydrofolate, thereby resulting in potent inhibition of TS enzyme activity. There are several lines of evidence to suggest that TS is a critical target in the clinical setting and they include: A) a close association has been found between the level of intracellular expression of TS and fluoropyrimidine sensitivity in that high intracellular levels of TS are generally associated with fluoropyrimidine insensitivity [4, 5]; B) a close association between the ability to inhibit the TS enzyme activity and ultimate clinical outcome has been observed in patients with advanced breast and gastric cancer [6, 7]; C) a number of positive clinical trials has now been reported showing that LV positively modulates 5-FU activity in patients with colorectal cancer in both the adjuvant and advanced disease settings [1, 8]. Using in vitro, in vivo and clinical model systems, it has been shown that treatment with LV markedly enhances the ability of the 5-FU metabolite FdUMP to specifically inhibit TS by providing the essential reduced folate substrate needed for prolonged enzyme inhibition. In the adjuvant setting, the addition of LV to 5-FU has resulted in an approximately 30% decrease in death rate in patients with Dukes B and C colon carcinoma [8], while in the advanced disease setting, an approximately twofold increase in overall response rate has been observed when compared to the use of single agent 5-FU [1], and D) recent clinical investigations using antifolate analogs that are potent and specific inhibitors of TS have shown promising clinical activity in patients with advanced colorectal cancer achieving response rates on the order of 30%-40% [3]. Taken together, these clinical observations support the central role of TS in defining the activity of fluoropyrimidine agents and suggest that improvements in targeting this central enzyme may result in significant clinical advances.

Recently, we reported on the clinical activity of the combination of 5-FU and LV in patients with advanced breast cancer [6]. Various biochemical endpoints were included in this trial in an attempt to provide correlative data with the clinical results. Specifically, TS enzyme levels were measured prior to and 24 h following 5-FU therapy in the tumors of patients with cutaneous disease. We observed that TS levels were increased nearly threefold in those biopsy specimens taken 24 h following 5-FU. Since the level of TS appears to be an important determinant of sensitivity to 5-FU, we postulated that this acute induction of TS might serve as an efficient mechanism by which malignant cells developed acute resistance to 5-FU. As shown in Table 1Go, similar observations have been made in several in vitro and in vivo preclinical experimental systems [4, 9–12]. In each case, a two- to fourfold increase in TS enzyme activity was noted approximately 24 h after exposure to 5-FU. Similar observations were made in human H630 colon carcinoma cells whereby exposure to 5-FU at a concentration of 1 µM resulted in a maximal three- to fourfold increase in the level of TS 24 h following drug exposure. Of note, simultaneous exposure to nongrowth inhibitory concentrations of {gamma}-interferon (IFN) completely abrogated the 5-FU-mediated induction of TS. This repression of TS by {alpha}-IFN resulted in an approximately 20-fold increased sensitization of these cells to the growth inhibitory effects of 5-FU. We reasoned, therefore, that a more complete understanding of the regulatory elements underlying the acute induction of TS following fluoropyrimidine exposure might provide the rational basis for alternative therapeutic strategies for the treatment of patients with fluoropyrimidine-responsive tumors.


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  		Table 1. Induction of thymidylate synthase
 
Our initial investigations demonstrated that the increased levels of TS protein following 5-FU exposure were not accompanied by an increase in the level of expression of TS mRNA, suggesting a post-transcriptional event underlying this induction of protein [13]. Subsequent experiments measuring both the stability and synthetic rate of TS in cells exposed to 5-FU revealed that the increased intracellular synthesis of TS was mediated by an enhanced translational efficiency of TS mRNA. To more directly investigate the regulation of TS mRNA translation, we subsequently employed an in vitro rabbit reticulocyte lysate translational system. We found that the addition of exogenous pure human TS protein nearly completely repressed the translation of human TS mRNA. In contrast, the addition of a different folate-dependent enzyme such as dihydrofolate reductase (DHFR) was unable to inhibit TS mRNA translation. In addition, the translational efficiencies of other unrelated mRNAs remained unaffected in the presence of the same human recombinant TS protein, providing support for the specificity of the translational repressive effects of TS protein on TS mRNA translation (Fig. 1Go) [14]. Taken together, this set of studies suggested that translation of human TS mRNA was regulated by its own protein product via a negative autoregulatory mechanism.



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  		Figure 1. Specific effect of TS on TS mRNA translation using the rabbit reticulocyte lysate in vitro translation system. Translation reactions containing rabbit reticulocyte lysate were incubated with either no exogenous mRNA (lane 1), or with 250 ng TS mRNA, 250 ng human chromogranin A mRNA, 250 ng yeast mRNA, 250 ng human folate receptor mRNA and 250 ng human preplacental lactogen mRNA, where indicated. Pure human recombinant TS protein (400 ng) was included in the reaction samples where indicated. In lane 4, DHFR (1,000 ng) was added to the reaction mix. Translation reactions were incubated at 37°C for 60 min, and protein products were resolved on a 15% SDS polyacrylamide gel. Adapted with permission [14].

 
While these initial studies suggested a direct interaction between TS protein and its own TS mRNA, RNA electrophoretic gel mobility shift assays were used to confirm a specific and high affinity interaction between TS mRNA and TS protein. Our working model of the translational autoregulatory control of TS is presented in Figure 2Go. Of note, while this form of regulation has been well described for various bacteriophage T4 proteins and a number of E. coli ribosomal proteins, this autoregulatory mechanism has not been previously described in eukaryotes. The intracellular level of TS is finely controlled by an autoregulatory mechanism whereby binding of the TS protein to its own TS mRNA inhibits translational efficiency resulting in a decrease in intracellular levels of TS. Two different regions on TS mRNA have now been identified that bind with high affinity, on the order of 1-2 nM, to TS [15]. The first site is located within the first 188 nucleotides (nts) and includes the translational start site contained within a putative stem-loop structure with the AUG start sequence in the loop aspect, while the second site is located within a 100-nt sequence in the protein-coding region. Most of the well-characterized cis-acting sequences mediating translational control identified thus far have been shown to reside in the 5'-untranslated region. For this reason, our initial efforts focused on the 5'-upstream binding site. Using a combination of mutant and deletional RNA sequences, we determined that a GCCAUG hexanucleotide sequence in the loop aspect is critical for protein recognition. Further studies are currently in progress to identify the optimal nucleotide sequences required for not only this 5'-upstream binding site but also for the protein-coding region binding site.



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  		Figure 2. Model of translational autoregulation of TS.

 
This RNA/protein interaction is markedly dependent upon the state of occupancy of the protein. When TS remains ligand-free, it retains complete RNA binding activity thereby leading to translational inhibition. However, when the protein is occupied by either of the physiologic substrates dUMP or 5,10-methylenetetrahydrofolate or the fluorinated nucleotide metabolite FdUMP, it can no longer bind to its mRNA, thereby relieving translational inhibition and resulting in increased intracellular levels of TS protein. Such a situation would exist in cells exposed to 5-FU. Thus, this model provides a rational mechanism for the acute induction of TS that arises following exposure to 5-FU. Presumably, this translational autoregulatory mechanism serves to control the level of TS during cellular proliferation through modulation of the protein interaction with RNA by its state of occupancy by physiologic substrates.

An enhanced understanding of the basic molecular elements underlying the interaction between TS and its own TS mRNA may serve as the foundation for the rational design and development of new therapeutic strategies. Several of the approaches that may stem from these studies are shown in Figure 3Go, and they include: A) the development of new inhibitors of TS enzymatic activity that would still allow for the TS protein/TS mRNA interaction. This would result in formation of a stable ternary complex of inhibitor, TS protein and TS mRNA resulting in inhibition of new TS protein synthesis; B) the development of "small molecules" mimicking the function of {gamma}-IFN that would serve to bind to the cis-acting binding sites on TS mRNA resulting in inhibition of new TS protein synthesis; C) the development of a small peptide or a small molecule mimicking the structure of the peptide that can be used to bind to TS mRNA, resulting in inhibition of new TS protein synthesis. Such an approach is currently being taken in anti-HIV drug development where small basic rev peptides that mimic the function of the native rev protein are being tested to inhibit HIV viral replication, and D) The development of either an RNA oligonucleotide or a small molecule mimicking the structure of the RNA that can be used to sequester and/or inhibit TS enzymatic function.



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  		Figure 3. Potential therapeutic strategies based on TS protein/TS mRNA interaction.

 
While the future clinical development of novel small molecules and novel therapeutic strategies awaits the results of these molecular studies examining the TS protein/TS mRNA interaction, our group has focused, over the past few years, on performing clinical trials that incorporate two well-characterized biochemical modulators of 5-FU, namely LV and {gamma}-IFN, that both result in an enhanced inhibition of the target enzyme TS [16, 17]. The schema for this trial is shown in Table 2Go and includes the use of IFN {alpha}-2a, given s.c. on days 1-7 that brackets a daily 5-day administration of LV 500 mg/m2 i.v., and 5-FU 370 mg/m2 on days 2 through 6. The 5 x 106 unit per m2 dose of {alpha}-IFN was determined to be optimal in a dose-seeking pilot study. In addition to its interaction at the biochemical and molecular levels, a pharmacokinetic interaction was noted between {alpha}-IFN and 5-FU in that concurrent use of {alpha}-IFN resulted in a 30% increase in plasma levels of 5-FU, primarily through a decrease in clearance of 5-FU. In a subsequent phase II clinical trial, 46 patients with advanced colorectal carcinoma were treated according to the schema outlined in Table 2Go. Eighty-nine percent of these patients had Eastern Cooperative Oncology Group performance status 0 or 1, and no patient had been previously treated with 5-FU. Dose-limiting toxicity consisted of mucositis and/or diarrhea in approximately 40% of patients at the grade III or IV level (NCI-Common Toxicity Criteria). The overall response rate in these patients was 54%, with four patients (9%) achieving a complete response. The median overall survival for the group was 16 months. Given these promising results, the National Surgical Adjuvant Breast and Bowel Project (C-05) has taken this regimen and compared it to a regimen containing only 5-FU and LV in patients with Dukes B and C colon carcinoma. This trial concluded its enrollment of approximately 2,000 patients in early 1994, and we await maturation of the clinical data to assess the contribution of {alpha}-IFN in a 5-FU/LV-based regimen for the treatment of patients with Dukes B and C colon carcinoma.


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  		Table 2. NCI regimen combining 5-FU with LV and interferon-{alpha}
 
Given the apparent clinical importance of TS as a therapeutic target, our laboratory has devoted significant efforts at developing sensitive and specific assays for its measurement in human tumor samples. To this end, a panel of monoclonal antibodies (mAbs) directed against human TS have now been developed, and we have found them to be highly sensitive and specific reagents [18]. A significant advantage of an antibody-directed approach for detection of TS over enzymatic-based assays is that a catalytically active enzyme is not required. Moreover, these mAbs may be used to quantitate the level of expression of TS in fresh, as well as in fixed, paraffin-embedded tissues. To begin to assess the potential role of TS as a significant prognostic indicator, we evaluated a series of primary tumor samples taken from patients entered into a National Surgical Adjuvant Breast and Bowel Project (R-01) rectal trial [19]. The sample size of 294 patients appeared to be representative of the entire group of patients entered into the investigation by virtue of the similarity of stage and survival of patients in the sample group versus the entire group. Patient samples were graded as high versus low TS levels. As shown in Figure 4Go, a highly significant correlation was found between TS levels and overall survival at five years in this patient population, such that patients with low TS levels had a greater overall survival when compared to those with high TS levels. Of importance, we observed that the prognostic significance of TS levels was independent of Dukes stage in that within each specific Dukes category, the levels of TS remained prognostic. These immunological assays have since been extended to studying patients with advanced gastrointestinal tumors in an attempt to identify the role of TS levels in predicting response to 5-FU-based regimens. These preliminary investigations have shown a significant correlation between the level of TS and ultimate responsiveness to fluoropyrimidine therapy, in that patients with low levels of TS tend to respond to fluoropyrimidine-containing regimens [20]. Studies to assess the prognostic significance of TS are now being extended to patients with early and advanced colon cancer, as well as to patients with breast and head and neck carcinomas, wherein 5-FU appears to have good clinical activity.



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  		Figure 4. Survival of patients with rectal cancer according to the level of TS expression. Reproduced with permission.

 
Hopefully, further investigations to elucidate the basic biology of malignant cells, the basic molecular mechanisms underlying the regulation of expression of the critical target enzyme TS and the mechanism(s) of interaction between 5-FU and other antineoplastic and/or biologic agents will lead to improved therapeutic strategies that incorporate currently available agents with novel agents or small molecules under development. Furthermore, the development of sensitive immunohistochemical-based assays for key clinical targets such as TS will hopefully lead to an enhanced ability to select specific patient populations most likely to derive benefit from currently available therapies.


   FOOTNOTES
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 Abstract
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From Advances in Cancer Treatment: The Chabner Symposium. STEM CELLS: 1996;14:41-46.


   REFERENCES
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 Abstract
 References
 

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