Drug resistance, comprised of redundant and interdependent biological pathways, is a major impediment to developing successful strategies for cancer therapy. New hope may reside in the RNA world.

Clinical success of drugs against individual targets known to cause drug resistance has been marginal, though the strategy is attractive for its simplicity. Building on clinical experience, new pharmacological drugs and modes of delivery are being designed to thwart genetic evasion and reduce toxic side effects.

Advances in methods for gene expression profiling are now converging with the exploding world of non-(protein)-coding RNAs (ncRNAs) and corresponding RNA interference (RNAi) technology. The therapeutic potential of RNAi to silence gene expression is already demonstrated for single genes and biological pathways involved in causing drug resistance.

Though exciting, realization of this technology will require solutions to its delivery and specificity as well as understanding new toxicities associated with long term use. In this regard, existing drugs can act as RNAi alternatives. One example is ONCONASE (ranpirnase), already in Phase IIIb confirmatory clinical trials for the treatment of unresectable malignant mesothelioma. Ranpirnase targets ncRNA(s) and is known to reverse multi-drug resistance in pre-clinical studies.¹ This article highlights the evolution in cancer treatments from standard chemotherapeutics to tumor-specific therapies and the strategies being employed to avoid development of resistance.

Cytotoxic drugs with optimal pharmacokinetics and tumor penetration can be effective against chemosensitive tumors such as breast cancer. Unfortunately, resistance can develop and present a major obstacle to further effective treatment. Many intracellular events contribute to drug resistance. The drug target may be mutated or amplified.

Intracellular levels of the drug may be decreased due to impeded entry into the cell or active ejection from the cell. In the latter case, multi-drug resistance often results from increased expression of ATP-binding cassette (ABC) transporter proteins that cause the efflux of various anticancer drugs from the cancer cell. The most widely studied of these is linked to a broad spectrum, ATP-dependent drug, efflux pump identified as P-glycoprotein (P-gp).² P-gp is encoded by the MDR1 (ABCB1) gene that has become a major target of strategies to reverse resistance to natural product drugs such as paclitaxel, doxorubicin, and vincristine.

Pharmacological strategies were sought to combat P-gp-associated resistance. Co-administration of drugs already in clinical use for other indications (e.g., verapamil, cyclosporine A, quinidine) or analogs of these first-generation drugs (e.g., dexverapamil, valspodar, cinchonine) achieved limited success.³ Now, new variations are specifically being developed for MDR reversal. One example, biricodar, is a novel compound that binds directly to P-gp blocking its efflux activity. While clinical data suggests that the pump inhibitors can re-sensitize some subsets of patients to chemotherapy, these pharmacological strategies have not generally been encouraging. Many of the compounds used to reverse MDR are limited by their toxicity.^3,4 Proposed solutions to deliver the drugs in various types of nanocapsules or biological therapies such as the P-gp-reversing antibody, MRK16, also have been disappointing. Studies to develop novel anticancer agents that are not substrates for the P-gp drug transporter but act on intracellular targets very sensitive to drugs continue to be developed. For instance, epothilones are potent anti-microtubule agents. They have demonstrated activity in the presence of taxane-resistance and appear promising in the treatment of prostate cancer.

Attempts to profile a database of drugs for negative correlations between the MDR phenotype and growth inhibitory action surprisingly revealed that some compounds exhibited a positive correlation; their toxicity was potentiated—not inhibited—in the presence of P-gp expression. One of these agents is a thiosemicarbazone (NSC73306). Hence, a new twist in combating MDR1 resistance proposes to exploit the presence of P-gp rather than block it.⁵

Targeted therapeutics
A breakthrough in cancer drug development was achieved when proteins critical to tumor development and survival were identified. This presented the possibility of transitioning from general cytotoxins to tumor-specific therapies. A partial list of these compounds follows:
• Trastuzumab is a humanized monoclonal antibody directed against the extracellular domain or the tyrosine kinase receptor HER2. It has clinical activity in breast cancer patients whose tumors over-express HER2.⁶
• Imatinib, a small molecule, has demonstrated amazing clinical results. It was designed to bind and block BCR-ABL, a constitutively-active tyrosine kinase that leads to unregulated growth and chronic myeloid leukemia.⁷
• Gefitnib and erlotinib are examples of epidermal growth factor receptor (EGFR; HER1)-associated tyrosine kinase inhibitors (EGFR TKIs).⁸
• Cetuximab is a monoclonal antibody EGFR TKI inhibitor.⁸

All of these new and exciting drugs are approved for treatment of metastatic cancer. All are shown to be susceptible to developing drug resistance. They target unique sites in a single protein and thus it should not be surprising if mutations that affect binding and function develop or are selected for during treatment. Currently, this is being combated by altering scheduling, dosing, and optimization of patient monitoring.

Second-generation TKIs (dasatinib) are designed to bind to multiple protein conformations. Recently, some of the TKIs were shown to reverse some P-gp- and MRP-1-mediated MDR. Thus, using them to reverse resistance associated with cytotoxic drugs is a possibility.⁹

Kinase inhibitors having more than one target (multi-kinase inhibitors) are generating excitement. Sunitinib malate is approved for imatinib-resistant gastrointestinal stromal tumors. Though resistance does occur, patients often respond to a second multi-kinase inhibitor.¹⁰

click images to enlarge ONCONASE-induced apoptosis in HL-60. The top photomicrograph contains ONCONASE-treated, hyper-condensed, fragmented chromatin showing apoptosis. The bottom photomicrograph contains untreated control. (Source: Alfacell Corporation)

One gene vs. many
In the previous examples of drug resistance to both standard and novel targeted cancer therapies, the attempts to restore tumor cell sensitivity focused on altering the function of a small number of proteins (e.g., P-gp) and TKIs. Although it is clear that one gene product can cause drug resistance (e.g., p53), that gene is likely linked to multiple genetic events. Thus, new strategies rely on methods for global genomic analysis. Gene expression profiling of cancer cells after treatment with anticancer drugs is identifying biological pathways that contribute to drug resistance.

The NF-κB pathway is one example. Activated NF-κB translocates from the cytosol to the nucleus where it binds to specific DNA sequences in the promoters of its target genes and induces their transcription. It is known from preclinical and clinical studies that inhibitors of NF-κB activation can overcome conventional drug resistance.¹¹ Recently, gene expression analysis demonstrated that an anticancer drug can regulate NF-κB target genes in a drug-resistant breast cancer cell line, directly demonstrating that the NF-κB pathway is involved in resistance to the drug. Thus, inhibitors of NF-κB may be able to prevent development of resistance to some drugs.

Likewise, a functional, genetic approach identified the PI3K survival pathway as a major determinant of trastuzumab-resistance in breast cancer.^12,13 PI3K pathway genes block activation of the apoptotic response. The identification of specific protein-coding genes in these pathways offers the potential of more selective targets. Consequently, efforts are underway to develop treatment strategies that target these specific signaling pathways or their downstream effectors. Additionally, identifying the expression products of genes associated with sensitivity to drugs could serve as prognostic indicators to monitor a patient’s response to therapy. New possibilities of combating resistance to these targeted therapeutics may be found in the RNA world.

Drug resistance and the RNA world
The phrase “The RNA World” is generally attributed to Walter Gilbert in referring to the role of catalytic RNAs in the origin of life on Earth. Interest in the RNA world exploded with the discovery of ncRNAs that exert their regulatory functions as RNA molecules. These ncRNAs play a role in a variety of cellular processes such as transcription and chromosome replication; RNA processing and translation; and protein degradation and translocation. Recently, an ncRNA has been implicated in causing drug resistance in human cancer cells by decreasing the propensity for apoptosis. This is especially interesting because many cancer cell lines are resistant to inhibitors of anti-apoptotic proteins that could otherwise trigger extensive cell death.¹⁴

One abundant class of ncRNA is comprised of small (about 22 nucleotides) microRNAs (miRNAs) that are processed from larger pre-miRNAs. These small RNA molecules play an important role in development, cell proliferation and apoptosis. Recently, miRNA expression was linked to chemosensitivity and resistance in cancer cell lines, implying that miRNA expression levels may provide clues to how cells respond to drugs. MicroRNAs are part of the RNAi post-transcriptional gene silencing phenomenon that occurs naturally in many organisms from yeast to humans. This naturally-occurring, cellular process is used by cells to down-regulate gene expression. Thus, it can be harnessed to prevent the expression of virtually any RNA target through administration of small interfering RNAs (siRNA) and related technologies. To this end, RNAi is being applied to decreasing the messenger RNA (mRNA) for various genes involved in drug resistance. The following partial list is compiled from numerous preclinical studies.

• MDR1(ABCB1) gene coding for P-gp, as well as other ABC transporters such as BCRP, can be inhibited by RNAi technology.
• RNAi can synergize with TKIs to enhance sensitivity to chemotherapeutics and targeted drugs.
• RNAi-mediated silencing of anti-apoptotic pathway genes (e.g., PI3K, NF-κB) enhances apoptosis and sensitivity of chemotherapeutics.

Clearly, preclinical studies of RNAi technology is already shown to reverse resistance to many of the gene products and pathways described in the previous sections.^15-18 Although this may seem to recapitulate many studies with sequence-specific antisense and ribozyme technologies, the hope is that RNAi technology will be more specific and efficient because it is an innate biological process that naturally suppresses gene expression. Moreover, as the role of miRNA in drug resistance is further delineated, the RNA molecules themselves will likely become targets for oligonucleotide-based therapies. Previous experience with traditional antisense based strategies is expected to pave the way for future clinical trials with RNAi technologies. However, successful application of RNAi to combating disease and drug resistance will depend on solving problems of delivery, stability and possible toxicities of “off-target effects” in long-term treatment.

Targeting ncRNA
The recent revelations into how RNA may be involved in causing and treating cancer are causing a paradigm shift toward RNA as a drug target. Almost all of the RNAi strategies are based on nucleic acid hybridization to mRNA of cancer-associated proteins or to ncRNAs themselves. Ranpirnase is known to target tRNA, one of the major cellular ncRNAs.¹⁹ It also decreases levels of miRNAs over-expressed in some cancers. Ranpirnase touches on many aspects of drug resistance as well as the RNA world, including:
• Exerting anti-tumor effects in the presence of P-gp-mediated drug resistance
• Decreasing NF-κB in leukemic T-cells
• Having activity not dependent on p53
• Targeting a ncRNA

Ranpirnase specifically cleaves its RNA target(s).¹⁹ This triggers cancer cell death that is associated with up-regulation of tumor suppressor genes, a decrease in oncogenic miRNAs and induction of biological pathways leading to apoptosis.²⁰ Ranpirnase synergizes with many types of drugs, acting independently of the p53 gene that confers resistance to most cytotoxic agents.²¹ It also decreases NF-κB. The blockade of NF-κB can overcome resistance to imatnib (tyrosine kinase inhibitor) and doxorubicin.²² One intracellular event, or a combination of these events, may explain how ranpirnase kills P-gp over-expressing neuroblastoma cells and reverses P-gp-mediated drug resistance in models of human colon cancer since its actions are independent of P-gp expression. More importantly, ranpirnase shows clinical activity in the face of resistance to standard chemotherapeutics.²³

Conclusion
New strategies for combating cancer and the accompanying resistance to these strategies are being developed in tandem. Hope resides in the RNA world because the small ncRNA molecules appear central to causing both cancer and sensitivity to drugs.

About the Authors
Susanna Rybak, worked at Harvard Medical School and the National Institutes of Health to develop RNase-based therapeutics from 1989 to 2005. She is continuing in this field by serving on the Scientific Advisory Board of Alfacell Corporation, a biopharmaceutical company focused on the discovery, development and commercialization of novel ribonuclease (RNase) therapeutics for cancer and other life-threatening diseases.

This article was published in Drug Discovery & Development magazine: Vol. 11, No. 4, April, 2008, pp. 18-24.