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10.1245/ASO.2003.07.518
Annals of Surgical Oncology 10:852-862 (2003)
© 2003 Society of Surgical Oncology
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NEW APPROACHES TO THE TREATMENT OF HEPATIC MALIGNANCIES

Overcoming Antiapoptotic Responses to Promote Chemosensitivity in Metastatic Colorectal Cancer to the Liver

James C. Cusack, Jr., MD

From the Division of Surgical Oncology, Massachusetts General Hospital, Boston, Massachusetts.

Correspondence: Address correspondence and reprint requests to: James C. Cusack, Jr., MD, Division of Surgical Oncology, Massachusetts General Hospital, 100 Blossom St., Cox 626, Boston, MA 02114; Fax: 617-724-3895; E-mail: jcusack{at}partners.org

ABSTRACT

Background: Metastatic colon cancer is highly resistant to chemotherapy. A variety of mechanisms by which cancer cells resist chemotherapy have been described including enhanced export of drug from cancer cells and alterations in drug metabolism. In addition, the response of cancer cells to genotoxic therapies may be diminished by acquired defects in either the response mechanisms to DNA damage or cell cycle regulatory pathways. Recently, attention has focused on mechanisms that are activated by treatment exposure and subsequently promote resistance by rescuing cancer cells from apoptosis. The objective of this review is to examine the role of antiapoptotic mechanisms of chemotherapy resistance and to determine the potential utility of therapeutic strategies that target these mechanisms.

Methods: To accomplish the objectives, a brief overview of mechanisms of chemotherapy resistance is provided. The concept of inducible chemotherapy resistance is introduced by examination of a specific antiapoptotic mechanism, mediated by the transcription factor, nuclear factor kappa B (NF-{kappa}B). The ability to use inhibitors of NF-{kappa}B to promote chemosensitivity is examined in vitro and in vivo.

Results: Inhibition of chemotherapy-induced NF-{kappa}B activation enhances apoptosis and augments chemotherapy sensitivity.

Conclusions: NF-{kappa}B inhibition may overcome cancer cell defense against apoptosis. Molecular therapies that target this resistance mechanism may be useful adjuncts to conventional chemotherapy.

Key Words: Chemotherapy resistance • Apoptosis • NF-{kappa}B • Colon cancer • Liver metastases

Most cancer cells remain resistant to chemotherapy and gamma irradiation.1–3 Efforts to overcome this resistance by increasing concentrations of cytotoxic drugs and using higher dosages of irradiation has failed to significantly improve the therapeutic response. To improve treatment response, researchers have strived to better understand the molecular basis of resistance. Findings from recent studies investigating a diverse array of cellular response mechanisms have contributed to a more comprehensive model of resistance that includes factors that either positively or negatively influence cancer cell death (apoptosis) response to treatment. In this article, we provide a brief overview of chemotherapy resistance and review some of the studies that have reshaped our understanding of drug resistance. In addition, we will examine a specific antiapoptotic mechanism, mediated by the transcription factor nuclear factor kappa B (NF-{kappa}B), as an example of how manipulation of the apoptotic response in cancer cells may contribute to enhance sensitivity to conventional chemotherapy.

MECHANISMS OF RESISTANCE TO ANTICANCER THERAPIES

Most solid tumors including colorectal cancer and hepatoma are frequently resistant to chemotherapy and irradiation.4 Cancer cells utilize a variety of mechanisms to protect against the damaging affects of anticancer therapies, some of which accompany the phenotypic changes characteristic of the tumorigenic transformation from normal to malignant cell. These intrinsic resistance mechanisms frequently represent the loss of normal physiologic responses or the acquisition of abnormal responses to physiologic stimuli that are encoded as errors in DNA. A second type of resistance occurs when established cancer cells are exposed to selection forces such as chemotherapy agents. In this setting, a resistance mechanism that imparts a survival advantage to a subclone of cancer cells leads to the expansion of cells with an acquired resistance mechanism. One further distinction that may be made is in regard to whether the resistance mechanism is manifested in the absence of the anticancer agent or requires exposure to treatment to become transiently active (inducible resistance). Taken together these resistance mechanisms impart a 50% to 80% likelihood of failure of solid malignancies to respond to treatment. Our ability to devise treatment strategies that overcome these resistance mechanisms is perhaps the most challenging obstacle to the successful treatment of patients with metastatic colorectal cancer and hepatoma.4

A variety of cellular processes are involved in drug resistance, including alterations in drug transport in and out of the cell, changes in the intracellular metabolic processing of the drug, and alterations in the target of drug therapy. Similarly, changes in the genes that regulate the cell cycle and the DNA damage repair mechanism similarly may result in the development of resistance to chemotherapy. Several specific cellular mechanisms that determine sensitivity of cancer cells to genotoxic therapies have recently been elucidated.5–8 For example, the mechanism by which the up-regulation of the multidrug resistance gene product (mdr1) renders cancer cells resistant to chemotherapy agents is by increasing the rate of efflux of these drugs from the cell.9 Still other mechanisms of resistance appear to affect a cancer cell’s ability to undergo apoptosis, the major mechanism by which chemotherapy and radiation induce the killing of tumor cells.10–12 Protection from the killing effects of anticancer therapies is exemplified by the high incidence of mutation in the p53 tumor suppressor gene in colorectal cancer. This genetic defect impairs p53-dependent responses to apoptotic stimuli, thereby promoting cancer cell survival and proliferation in cancer cells undergoing treatment.13 Recent attention has focused on the mechanism of inducible chemotherapy resistance, a process whereby exposure of tumor cells to cancer therapy leads to the transient activation of a cellular defense mechanism that protects cancer cells against the damaging effects of anticancer therapies.9 The reversible nature of this survival response presents new opportunities for molecularly targeted cancer therapies. This later mechanism will be presented in detail in this article.

At the cellular level, alterations in the transport of drugs in and out of the cell can dramatically alter the ability of the drug to reach its target or impart the desired response. Intrinsic and acquired multidrug resistance (MDR) in many human cancers is attributable to the expression of the multidrug transporter P-glycoprotein (Pgp), encoded by the mdr1 gene. In chemotherapy-naïve colorectal and renal cell cancers, constitutive Pgp expression, acquired through the genetic changes that accompany the tumorigenesis process, appears to have prognostic significance and negatively impacts survival.14 In other malignancies, including leukemias, lymphomas, myeloma, and breast and ovarian carcinomas, Pgp expression is acquired through the course of repeated chemotherapy exposure.14 In these cases, Pgp expression results in a progressive decrease in intracellular drug concentrations while serum drug levels remain constant. Treatment response to a given dosage is thereby diminished by a loss in drug sensitivity and decreased tumoricidal effectiveness.

CHEMOTHERAPY RESISTANCE MEDIATED BY ALTERED DRUG METABOLISM

Differences in drug metabolism lead to significant variation in the concentration of drug in both plasma and tissues and may result in a wide range of tumoricidal and toxic effects. Phenotypic groupings of metabolic enzymes based on functional variations in host metabolic rate are currently determined by the administration of a probe drug and measurement of metabolites in plasma or urine.15 Cytochrome P-450 is perhaps one of the best characterized drug-metabolizing enzymes. Enzymes in the CYP3A subfamily of cytochrome P-450 play a role in the metabolism of many drugs, including epipodophyllotoxins, ifosfamide, tamoxifen, paclitaxel, and vinca alkaloids.16 Subtle alterations in these enzymes account for some of the interindividual variations in drug metabolism that in part affect toxicity and tumor response.

Treatment of patients with advanced colorectal cancer typically involves a 5-fluorouracil (5-FU)-based chemotherapy regimen, such as 5-FU/levamisole or 5-FU/folinic acid. Recently, CPT-11 received Food and Drug Administration approval for use as an adjunct to 5-FU-based regimens for the treatment of patients with metastatic colorectal cancer. However, many patients continue to experience progression of disease on these treatments, due in part to intrinsic and/or acquired tumor resistance. Although expression of the mdr1 gene may potentially alter drug resistance in some cancers, chemotherapy resistance in most colorectal cancers likely involves other mechanisms other than overexpression of the mdr1 gene. DeAngelis et al. detected Pgp in 44% (15/34) of the colorectal cancers sampled and in 100% (13/13) of the normal colonic mucosas (P = .0005), with highest levels of expression seen in normal mucosa.17 In addition, agents used to treat patients with colorectal cancer such as the topoisomerase-1 inhibitor CPT-11 (active metabolite is SN-38) have been found to have sustained activity against chemotherapy-resistant colon cancer cell lines, including those having the MDR phenotype.4 Using mass spectrometry and microsequencing, Sinha et al. have reported two potential genes that were overexpressed in resistant colorectal tumor cell lines and may be involved in colorectal cancer chemotherapy resistance, adenine phosphoribosyltransferase and breast cancer specific gene 1 (BCSG1).18 Other potentially relevant mechanisms of resistance to 5-FU-based chemotherapy suggested by Mini et al. include the increased expression of thymidylate synthase and the decreased expression of folylpolyglutamate synthetase expression. In studies to assess the expression of these genes in colorectal tumors from patients undergoing surgery and postoperative chemotherapy, compared with normal colonic mucosa, they found that overall and disease-free survival suggest an inverse relationship between the level of tumor thymidylate synthase and folylpolyglutamate synthetase expression and clinical prognosis.19 Due to the complexity of the mechanisms involved, it is likely that several genes will emerge as important mediators of chemotherapy resistance in colorectal cancer.

APOPTOSIS: THE ULTIMATE OBJECTIVE OF CYTOTOXIC ANTICANCER THERAPIES

While delivery of a drug to its target is critical to its therapeutic effect, the ability of a cytotoxic drug to successfully induce cancer cell death generally requires intact intracellular transduction signaling pathways to initiate the cellular program that commits a cancer cell to cell death. Kerr et al. first described this mechanism of controlled cell deletion or apoptosis in 1972, as a highly organized phenomenon that may be initiated or inhibited by various physiological and pathological stimuli.20 Over the past 5 years, intensive effort has been directed toward furthering our understanding of the role of programmed cell death in cancer to develop more effective therapies. Using the paradigm of programmed cell death, resistance to conventional anticancer therapies can be distilled down to the proapoptotic and antiapoptotic responses that are induced by these treatments. Novel cancer treatment strategies that promote apoptosis and block factors that inhibit apoptosis may dramatically augment cancer sensitivity to conventional therapies.

THE ROLE OF APOPTOSIS IN PRIMARY AND METASTATIC MALIGNANCIES OF THE LIVER

Genetic aberrations, arising spontaneously or resulting from exposure to genotoxic agents, may produce a mutation or deletion in tumor suppressor genes or activation of proto-oncogenes. The composite genetic abnormalities that result during tumorigenesis lead to the expression of the malignant phenotype in both primary and secondary liver cancers. The multistep tumorigenesis model, proposed by Vogelstein and Fearon, describes this step-wise transformation from the benign polyp to the malignant phenotype that results from the accumulation of genetic errors in colorectal cancer.21,22 A similar multistep process has been observed in a hepatocarcinogensis model in rats.23 In each of these models, it appears that tumorigenesis is associated with not only an increase in cell replication but also a change in the rate of cell death.While an increased rate of replication is present in all stages of tumorigenesis, the level of apoptosis does not appear to be a static feature of tumorigenesis. Down-regulation of apoptosis appears to be critical to the early tumorigenic changes of hepatocellular cancer development.23 Nongenotoxic factors such as ethanol, cytotoxic and inflammatory events, steroid hormones, and some drugs, as well as genotoxic agents such as certain mycotoxins and hepatitis B virus protect preneoplastic foci in the liver from apoptosis, allowing these foci to develop into hepatocellular adenoma, and ultimately hepatocellular carcinoma.23 It is likely that a variety of cancer cell survival factors decrease the level of apoptosis at specific stages of tumorigenesis and are critical to the development of malignant tumors.

EFFECT OF MUTATIONS IN TUMOR SUPPRESSOR GENES AND ONCOGENES ON APOPTOSIS

Increasing evidence suggests that several oncogene products may affect tumorigenesis, in part, through the antiapoptotic effect these genetic factors have on cancer cell survival.24 The antiapoptotic function of transforming genetic errors has been examined at the molecular level in the context of several genetic abnormalities, including mutations of the p53 tumor suppressor gene and alterations of the Bcl-2 family of genes. P53 mutations, usually associated with loss of the wild-type allele, are found in 25–50% of hepatocellular cancers and have been found to have a positive correlation with both increasing histological grade and early recurrence.25,26 Similarly, as many as 60–80% of colorectal cancers may have a mutated or deleted p53 gene.22 Enhanced level of expression of the wild-type p53 protein, following genotoxic injury typically functions to induce cell cycle arrest to repair DNA damage or under certain conditions to induce apoptosis.27,28 The lack of wild-type p53 function will therefore result in the expansion of preneo-plastic clones, as may be the case in chronic liver disease, by failing to induce cell cycle arrest to correct errors in DNA replication. In the case of hepatoma, these events may signal the initial stages of carcinogenesis.25

Studies by Symonds et al. found that the induction of aggressive choroid plexus tumors, in response to a weak oncogenic event (SV40 T antigen fragment), corresponds to a decreased level of p53-dependent apoptosis.29 Tumor growth in this model was associated with a reduced level of apoptosis rather than increased cell proliferation. The results of this study indicate that (1) wild-type p53 functions, in response to abnormal proliferation, by suppressing tumor growth through induction of apoptosis of abnormally proliferating cells and (2) p53-dependent apoptosis in response to oncogenic events is a critical regulator of tumorigenesis. While the presence of wild-type p53 does not necessarily indicate the capacity of a cell to undergo apoptosis, the absence of wild-type p53 resulted in decreased levels of apoptosis in the presence of oncogenic triggers among some cell types. These data imply that the late acquisition of a single additional "hit", such as a p53 mutation/deletion, in the multistep progression of several human cancers, may account for both loss of proliferation control and inhibition of apoptosis, characteristic of the malignant phenotype. This later feature suggests that correction of the genetic alteration, for example, transfer of the wild-type p53 gene to p53 mutant cancer cells, would have potential therapeutic benefit by correcting the genetic abnormality and in this case restoring the p53-dependent cell cycle arrest and apoptotic responses.

THE AFFECTS OF CONVENTIONAL ANTICANCER THERAPY ON APOPTOSIS

Roth and Cristiano have advocated the use of gene therapy in combination with chemotherapy and irradiation to augment the efficacy of conventional therapies.30 While this approach has the advantages of combining reconstitution of the apoptotic response mechanism with exposure to apoptotic stimuli, this approach to date rarely results in cures in animal tumor models. One possible explanation for the limited success of this approach is the dependence of the apoptotic response on functional downstream effector pathways. Friedman et al. recently demonstrated that inducible p53 expression in p53 null Hep3B hepatoma cells failed to induce growth arrest or apoptosis when combined with cisplatin despite p21 (waf 1) induction.31 In these experiments, the presence of a nonfunctional retinoblastoma protein and the failure to induce bax mRNA transcription likely contributed to failed growth arrest and apoptosis, respectively. One additional obstacle to the success of combined chemotherapy and gene replacement therapy, however, is the recent finding that inducible antiapoptotic factors discussed previously are activated in response to chemotherapy and irradiation, in parallel to the induction of the apoptotic response mechanism. Importantly, to be effective, anticancer therapies require not only an intact system of downstream regulators but also inactivation of inhibitory pathways that may negatively influence the apoptotic response mechanism.

NF-{kappa}B ACTIVATION INDUCED BY CANCER THERAPIES BLOCKS THE INDUCTION OF APOPTOSIS

Most cancer therapeutics function by killing cells through the induction of the apoptotic pathway. In fact, it has recently been recognized that resistance to the induction of the apoptotic response is a principle mechanism by which cancer cells protect against cell killing.10 Recently, investigators have begun to unravel an inducible mechanism by which cancer cells are protected against the damaging effects of anticancer treatments. Wang et al. reported that the activation of NF-{kappa}B by tumor necrosis factor-alpha (TNF-{alpha}), ionizing radiation, and the cancer chemotherapeutic compound daunorubicin leads to an inhibition of the apoptotic response induced by these stimuli in fibrosarcoma cells.32 Similar results were obtained by other investigators33–35 relative to TNF-{alpha}. These findings suggest that apoptotic stimuli such as chemotherapy, radiation, and TNF initiate two distinct signaling pathways: one that leads to activation of apoptosis and a parallel pathway that leads to NF-{kappa}B activation and the induction of a cell survival response that inhibits apoptosis (Fig. 1).32,36–40 The mechanism by which chemotherapy activates NF-{kappa}B is presently unknown and is the focus of ongoing investigation. In contrast, the mechanism whereby NF-{kappa}B suppresses apoptosis is better understood and involves the induction of expression of genes that block the caspase cascade.36 We have previously shown that inhibition of inducible NF-{kappa}B activation by transient expression of the selective NF-{kappa}B inhibitor, super-repressor I{kappa}B{alpha}, leads to a dramatic improvement in the killing response of tumor cells to apoptotic stimuli.41 Based on these preliminary studies, it was concluded that the apoptotic response to conventional chemotherapy and irradiation may be significantly augmented by the inhibition of NF-{kappa}B activation in resistant cancer cells. The ability of activated NF-{kappa}B to mediate an antiapoptotic response following exposure of cancer cells to apoptotic stimuli has been demonstrated in a wide variety of human cancer cell lines.41–43



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  		FIG. 1. Inhibition of apoptosis by activation of nuclear factor kappa B (NF-{kappa}B) in response to chemotherapy, ionizing irradiation, and tumor necrosis factor-alpha is an inducible mechanism by which cancer cells may resist the cytotoxic effects of anticancer therapy. Activation of NF-{kappa}B results in expression of antiapoptotic proteins that block apoptosis through inhibition of the caspase cascade as well as through a caspase-independent mechanism that has not yet been defined.

 
SN38 INDUCES ACTIVATION OF NF-{kappa}B IN A VARIETY OF HUMAN COLORECTAL CANCER CELL LINES

Anticancer agents, traditionally used to treat patients with metastatic colorectal cancer, including 5-FU and mitomycin C, have been found to only weakly induce NF-{kappa}B activation in a variety of colorectal cancer cell lines tested (Cusack, unpublished data, 2003). However, in preclinical studies, a wide variety of human colorectal cancer cell lines were found to undergo chemotherapy-induced activation of NF-{kappa}B, including cells expressing mutated p53 (WiDR, KM12L4, KM12SM, SW480, SW620), K-ras oncogene (LOVO, HCT116, SW480, SW620), and the antiapoptotic factor Bcl-2 (KM12L4, SW480) (Fig. 2A). In this experiment, the active metabolite of CPT-11 (SN38) was found to activate NF-{kappa}B in 10 of 11 colorectal cancer cell lines tested. Importantly, in all cases in which NF-{kappa}B activation was induced by SN38, inhibition of activation was facilitated by pretreatment with super-repressor I{kappa}B{alpha} but not the control vector (Fig. 2A). These findings are consistent with our observations in a variety of different cancer subtypes including pancreatic cancer, sarcoma, and breast cancer in which SN38 was found to be a very potent and consistent inducer of NF-{kappa}B activation. In addition, relative to a variety of different anticancer agents tested, the level of inducible NF-{kappa}B activation following treatment with SN38 is surpassed only by TNF-{alpha} (data not shown).



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  		FIG. 2. (A) The electrophoresis mobility shift assay was used to evaluate nuclear factor kappa B (NF-{kappa}B) activation induced by 1 µg/mL SN38 (the active metabolite of CPT-11) in human colorectal and breast (MCF-7) cancer cell lines. Chemotherapy-induced activation of NF-{kappa}B was observed in 11 of 12 cancer cell lines tested, suggesting that NF-{kappa}B activation is induced by SN38 in most colorectal cancer cell lines. Positive control (+) was KM12L4 cells treated for 2 hours with 10 ng/mL tumor necrosis factor-alpha (a potent activator of NF-{kappa}B). (B) Effect of NF-{kappa}B inhibition on human cancer cells treated with different concentrations of SN38. Cells were infected with adenovirus expressing the super-repressor I{kappa}B{alpha} (SR-I{kappa}B{alpha}) or control virus (CMV) 24 hours before drug treatment. Cell counts obtained at 96 hours following drug treatment are reported as the mean of triplicate cultures; bars, SD. [From Cusack JC, Liu R, Baldwin AS. Inducible chemoresistance to 7-ethyl-10-[4-(1-piperidino]-carbonyloxycamptothecin (CPT-11) in colorectal cancer cells and a xenograft model is overcome by inhibition of nuclear factor-{kappa}B activation. Cancer Res 2000;60:2323–30; modified with permission].

 
To evaluate the role of SN38-induced NF-{kappa}B activation on the chemosensitivity of a variety of resistant colon cancer cell lines, we pretreated these cells with the super-repressor I{kappa}B{alpha} (Fig. 2B). In all cell lines tested, sensitivity to SN38 was markedly enhanced by transient inhibition of NF-{kappa}B activation. Enhanced cytotoxicity responses attained through inhibition of NF-{kappa}B were found to be independent of the status of p53, K-ras, or Bcl-2 expression. Furthermore, enhanced chemosensitivity following pretreatment with super-repressor I{kappa}B{alpha} was observed at all concentrations of SN38 tested (Fig. 2B). This has important clinical relevance, in that dose-limiting toxicity frequently limits the clinically tolerated chemotherapy dosage to subtherapeutic levels. However, these findings suggest that at the lower tolerated levels of chemotherapy attained in patients, enhanced chemosensitivity may be achieved by NF-{kappa}B inhibition in those cells, thereby lowering the therapeutic threshold of the drug. Although the ability to predict sensitivity of a patient’s tumor to a specific chemotherapy agent is limited, the observed ability to augment the sensitivity of the variety of cell lines tested suggests that application of this combination therapy approach may have a broad impact on colorectal cancer patients receiving CPT-11.

DOSE INTENSIFICATION USING SERIAL ADMINISTRATION OF COMBINED CPT-11 AND NF-{kappa}B INHIBITION LEADS TO COMPLETE TUMOR ERADICATION IN A COLORECTAL CANCER XENOGRAFT MODEL

The findings of constitutive NF-{kappa}B in some forms of solid malignancy such as pancreatic cancer44 and soluble malignancies such as lymphoma or multiple myeloma45 would suggest that NF-{kappa}B inhibitors used as monotherapy may have potential use in the treatment of these malignancies. While early studies suggest that monotherapy may be effective in the soluble malignancies,46 our findings suggest that inhibition of inducible NF-{kappa}B activation when combined with chemotherapy provides the greatest tumoricidal response in solid malignancies. By inhibiting the inducible survival response, mediated by inducible NF-{kappa}B activation at each chemotherapy administration, the apoptotic response to CPT-11 treatment was optimized in vivo (Fig. 3). These data indicate that inhibition of NF-{kappa}B augments the apoptotic response to chemotherapy in a synergistic fashion. Thus, we conclude from these studies that optimal anticancer effects are obtained when each chemotherapy treatment is paired with an inhibitor of NF-{kappa}B. These preclinical studies therefore provide a rational basis for effective application of combination therapy that optimizes the apoptotic response and maximizes tumoricidal kill.



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  		FIG. 3. (A) Effect of nuclear factor kappa B (NF-{kappa}B) inhibition on LOVO cells treated with SN38. Cells were pretreated with adenovirus control vector (CMV) or adenovirus expressing the super-repressor I{kappa}B{alpha} (AdIkBa) 24 hours before drug treatment. Cell counts were obtained at 96 hours after drug treatment. (B) Electrophoretic mobility shift assay of nuclear protein extracts from LOVO tumors after treatment with the super-repressor I{kappa}B{alpha} and CPT-11 was used to evaluate the ability of Ad.CMV.I{kappa}B{alpha} to inhibit NF-{kappa}B activation in vivo. Tumors were treated with a single intratumoral injection of Ad.CMV.I{kappa}B{alpha} (I{kappa}B{alpha}) or control vector CMV. Animals were treated with CPT-11 24 hours after adenovirus injection and harvested at time 0 (24 hours after virus treatment), 1, 2, and 6 hours after drug treatment. Positive control is WiDr cells treated with SN38 at 2 hours. (C)Tumoricidal response of LOVO tumors to CPT-11 administered in combination with the adenovirus vector expressing the super-repressor I{kappa}B{alpha} (Ad.CMV.I{kappa}B{alpha}) compared with control adenovirus (Ad.CMV3), or vehicle alone. Adenovirus was administered as a weekly intratumoral injection of 1 x 1010 pfu/200 µL x 3 weeks. CPT-11 (33 mg/kg) was administered intravenously every 4 days during the 20-day treatment period. PBS was administered intravenously as a control. Tumor diameter along two orthogonal axes was recorded every other day. Tumor volume was recorded as the mean ± SE (bars) for each treatment group (n = 15–19). [From Cusack JC, Liu R, Baldwin AS. Inducible chemoresistance to 7-ethyl-10-[4-(1-piperidino]-carbonyloxycamptothecin (CPT-11) in colorectal cancer cells and a xenograft model is overcome by inhibition of nuclear factor-{kappa}B activation. Cancer Res 2000;60:2323–30; modified with permission].

 
PROTEASOME INHIBITION AUGMENTS CHEMOSENSITIVITY THROUGH INHIBITION OF CHEMOTHERAPY-INDUCED ACTIVATION OF NF-{kappa}B

The development of clinical-grade small molecular compounds that inhibit NF-{kappa}B has shifted attention in our laboratory from selective gene therapy-based approaches to nonselective small molecule methods of inhibiting NF-{kappa}B activation in combination therapy. Small molecule inhibitors of proteasome function represent one class of drugs that we have explored in depth in our laboratory for this purpose. These drugs that represent a novel class of anticancer agents called proteasome inhibitors that yield effective anticancer responses both in vitro and in vivo have been introduced to the treatment of malignancy.47,48 These drugs target the ubiquitin-proteasome pathway (specifically the 26S proteasome), the principle mechanism by which cellular proteins, including ubiquitinated I{kappa}B{alpha}, are degraded49–51 (Fig. 4). Inhibition of the ubiquitin-proteasome pathway results in dysregulation of cellular proteins involved in cell cycle control, promotion of tumor growth, and induction of apoptosis.47 The dipeptide boronic acid analogue PS-341, (VELCADETM) developed by Millennium Pharmaceuticals, Cambridge, MA, and used in these experiments, inhibits the chymotryptic activity of the proteasome in a potent, reversible, and selective manner.47 Proteasome inhibition using this compound has demonstrated significant cytotoxic activity against a range of human tumor cell lines that comprise the National Cancer Institute in vitro screen and is well tolerated in preclinical animal studies and in phase I clinical trials.47,48 The ability of proteasome inhibitors to regulate NF-{kappa}B activation has led many to speculate that these new compounds may have important clinical implications in the management of both inflammatory disease and malignancy.52



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  		FIG. 4. Proteasome inhibition provides a nonselective method for inhibiting nuclear factor kappa B (NF-{kappa}B) activation through the stabilization of the NF-{kappa}B inhibitory protein I{kappa}B{alpha}. A variety of cellular processes including cell cycle regulation are impacted by this approach. Following exposure to an apoptotic stimulus, I{kappa}B{alpha} is phosphorylated and subsequently polyubiquitinated. Under physiologic conditions the polyubiquitinated I{kappa}B{alpha} becomes dissociated from NF-{kappa}B, thereby exposing the nuclear localization signals on the NF-{kappa}B p50/p65 heterodimer. Following nuclear localization, NF-{kappa}B binds to DNA leading to the expression of a variety of genes, including those involved in the regulation of inflammation, and apoptosis.

 
To determine if pretreatment of cancer cells with PS-341 can effectively block the activation of NF-{kappa}B that is induced by exposure to SN38/CPT-11 and augment the sensitivity of tumor cells to chemotherapy, preclinical studies were conducted in colon cancer models. Similar to the findings we reported using the super-repressor I{kappa}B{alpha},41,53 proteasome inhibition using PS-341 potently blocked the early activation of NF-{kappa}B at 2 hours following chemotherapy treatment (Fig. 5B).54 In addition, combining proteasome inhibition with chemotherapy treatment resulted in a significant anticancer effect in vitro (Fig. 5A) and in vivo (Fig. 5C) compared with treatment with single agent alone.54 Terminal deoxynucleotide transferase dUTP nick end labeling assays, performed to determine if this increased anticancer effect from combination treatment was due in part to regulation of apoptosis, demonstrated markedly increased levels of apoptosis compared with single agent treatment alone (data not shown). This observed response is comparable to the response observed when NF-{kappa}B was selectively inhibited using the super-repressor I{kappa}B{alpha} in our previous reports.41,53



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  		FIG. 5. (A) The effect of proteasome inhibition (1 µM x 1 hour) on the sensitivity of LOVO colorectal cancer cells to treatment with SN38 was determined. Cell counts were obtained 96 hours after drug treatment. (B) The electrophoretic mobility shift assay was used to evaluate the effect of PS-341 (1 µM) treatment on the activation of nuclear factor kappa B (NF-{kappa}B) induced by treatment with SN-38 (1 µg/mL) in human colorectal cancer cells. Cell cultures were treated with PS-341 or mock control for 1 hour, followed 3 hours later by treatment with SN38. Cells were harvested and assayed for nuclear translocation of NF-{kappa}B at 1, 2, and 6 hours after chemotherapy treatment. Positive control (+) was KM12L4 cells treated for 2 hours with 10 ng/mL tumor necrosis factor-alpha (a potent activator of NF-{kappa}B). (C) The effects of combination therapy using the proteasome inhibitor PS-341 and CPT-11 were assessed in a xenograft model (n = 8–10/group). One-centimeter diameter LOVO tumors were grown in the flanks of nude mice. The tumoricidal effects of systemic treatment were assessed following pretreatment with PS-341 (1 mg/kg intravenous bolus injection) or vehicle alone, followed by intravenous bolus administration of CPT-11 (33 mg/kg) or vehicle. Treatment was administered 2x per week and tumor diameter along two orthogonal axes was recorded every other day. [From Cusack JC, Liu R, Houston M, et al. Enhanced chemosensitivity to CPT-11 with proteasome inhibitor PS-341: implications for systemic nuclear factor-{kappa}B inhibition. Cancer Res 2001;61:3535–40; modified with permission].

 
MECHANISMS OF ACTION IN RESPONSE TO PROTEASOME INHIBITION USING PS-341

Our findings suggest that the enhanced anticancer effects, observed when PS-341 is combined with chemotherapy, are due in part to inhibition of inducible NF-{kappa}B activation. However, other cell cycle regulatory processes and apoptotic response mechanisms impacted by proteasome inhibition may play a contributing role. For example, enhanced sensitivity to SN38/CPT-11 when combined with proteasome inhibition may in part result from the impaired metabolism and longer half-life of the topoisomerase 1-camptothecin complex that results when proteasome function is inhibited.55 Proteasome inhibition results in prolonged binding of the camptothecin to the topoisomerase-1 enzyme in a complex that under physiologic conditions would be degraded by the proteasome. Treatment with PS-341 effectively increases the duration of activity of the SN38/CPT-11. An additional mechanism by which proteasome inhibition may augment drug sensitivity is suggested by the findings that cell cycle regulatory proteins p21, p27, and p53 are stabilized by chemotherapy or PS-341 treatment (data not shown). Stabilization of these key cell cycle regulatory proteins has clear potential to impact the antitumor efficacy of these molecules alone or in combination. However, the ability to enhance chemosensitivity by proteasome inhibition has been observed in cells expressing wild-type p53 (CCD841, LOVO), mutant p53 (KM12L4, WiDR), and Bcl-2 (KM12L4). These findings are consistent with our previous report in which we found that inhibition of chemotherapy-induced NF-{kappa}B activation using the super-repressor I{kappa}B{alpha} occurred independent of the functional status of p53 and other regulators of apoptosis in colorectal cancer cell lines.

ANTICANCER THERAPY USING COMBINED PROAPOPTOTIC THERAPY WITH INHIBITION OF ANTIAPOPTOTIC FACTORS

The selection of chemotherapeutic agents and combination therapies that do not induce antiapoptotic defense mechanisms may prove to be an important consideration when developing new strategies to overcome chemotherapy resistance. Our studies using adenovirus-mediated transfer of the super-repressor I{kappa}B{alpha} to selectively block NF-{kappa}B have demonstrated the ability to markedly increase chemotherapy sensitivity through the induction of apoptosis both in vitro and in vivo.42,43 The ability of proteasome inhibitors to similarly promote enhanced chemotherapy sensitivity and enhanced apoptosis in preclinical models through the nonselective inhibition of NF-{kappa}B activation facilitates the evaluation of this novel treatment strategy in patients with chemotherapy resistant malignancies. Clinical trials are currently underway at the Massachusetts General Hospital and the University of North Carolina at Chapel Hill to evaluate the safety and potential efficacy of this combination therapy approach. In one study, the proteasome inhibitor PS-341 is combined with the topoisomerase I inhibitor CPT-11 to treat patients with refractory colorectal cancer. In a second study, PS-341 is combined with gemcitabine to treat patients with refractory pancreatic or lung cancer. The results from these initial clinical studies will likely shed light on the particular role NF-{kappa}B plays in these human cancers.

The evaluation of this combination therapy using selective small molecule inhibitors of NF-{kappa}B, IkappaB kinase inhibitor, is currently underway in our laboratory. Selective NF-{kappa}B inhibitors offer the theoretical advantage of inducing fewer deleterious side effects while specifically targeting both constitutive and inducible NF-{kappa}B activation. Such compounds may play a role not only as an adjuvant to chemotherapy but also in cancer prevention in high-risk patients in which NF-{kappa}B activation provides a critical survival function for precancerous neoplasms. The mechanisms by which NF-{kappa}B protects against apoptotic stimuli remain to a large extent elusive. However, the explosion of knowledge in both this transduction signaling pathway and others involved in chemotherapy resistance provides new targets for molecular therapeutics that may some day lead to the eradication of chemotherapy resistance.

FOOTNOTES

Inducible chemotherapy resistance is mediated by the transcription factor nuclear factor kappa B (NF-{kappa}B). Inhibitors of NF-{kappa}B administered in combination with chemotherapy augment apoptosis and enhance tumoricidal response. Combination treatment strategies that target antiapoptotic response mechanisms warrant further evaluation in multidrug clinical trials.

Received for publication July 18, 2002. Accepted for publication July 29, 2003.

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