Given the central role of oncogenes in driving tumorigenesis, oncogene products are a logical target in cancer therapy. Several studies have shown that oncogene inactivation in transgenic mice which have been engineered to express that particular oncogene will undergo tumor regression (1). Inactivation of a single oncogene by pharmacological agents or by adoptive T cell therapy can result in cancer regression, further supporting the hypothesis that cancers may be "addicted" to certain oncogenes to maintain cancer cell survival and proliferation (1). However, therapy that targets a single oncogene would not necessarily halt tumor growth: reducing the malignant phenotype and reversing the process of tumorigenesis is just one possible outcome of inactivating an oncogene. Other potential outcomes are outlined below:
In this scenario, the targeted oncogene may only be essential for the initiation of tumorigenesis. Since cancer develops through a multistage process, once initiated cells gain additional mutations and have progressed to a further stage, the deactivation of the initial oncogene may have no effect as tumor cells would no longer require its activity.
Oncogene inactivation may only suppress one or more neoplastic properties of the tumor cells but not all of them. For example, if an oncogene responsible for the metastatic properties of a tumor is inactivated, the tumor cells may lose their invasive potential, however, the tumor remains.
In reversion, the tumor cells may revert back to the status of a normal cell. For example, if a single oncogene was involved in all of a tumor cell's cancerous properties, such as metastatic potential, uncontrolled proliferation, and angiogenesis, the inactivation of this single oncogene would theoretically revert the cell back to its precancerous state.
Arrest, Death and Differentiation
Finally, oncogene inactivation may allow tumor cells to regain sufficient normal function such that regulatory mechanisms could induce cell cycle arrest, differentiation and apoptosis.
Transgenic models were developed to investigate how different oncogenes are initiated and sustained (2). The tetracycline regulatory system is often used in these transgenic models to conditionally activate oncogenes (3). The dimerized form of tetracycline transactivator (tTA) has two tetracycline or doxycycline (Dox) binding sites and a tetracycline operator (tetO) DNA binding site. When not bound to Dox, dimerized tTA binds to the seven tetO concatamer sequence and activates oncogene transcription. When bound to Dox, dimerized tTA changes conformation and loses its seven tetO binding affinity, and the oncogene is inactivated. Thus, the engineered oncogene can be activated and inactivated. Using this system, the effects of the inactivation of different oncogenes in different cancer types can be accessed. This led to the discovery that the inactivation of MYC in lymphoma and leukemia results in cell cycle arrest, differentiation and apoptosis by the tetracycline-dependent regulatory system.
As discussed earlier in this section, cancers are caused by multiple genetic changes such as oncogene activation and tumor suppressor gene inactivation. Also, cancer cells frequently have unstable genetic structures. A cell line that was previously dependent on the activation of a single oncogene may mutate rapidly into a cell line utilizing many different oncogenes. Therefore, inactivation of only one oncogene may not be enough to sustain tumor regression. However, many experimental results confirm that the single inactivation of some oncogenes can lead to tumor regression. For example, the inactivation of K-ras was found to induce apoptotic regression of lung adenocarcinomas in the absence of tumor suppressor genes p53 and Arf (4). Furthermore, the inactivation of c-MYC was found to induce rapid regression via vascular degeneration and apoptosis in pancreatic tumors caused by both c-MYC and BCKxL oncogenes (5). These findings suggest that the single inactivation of important oncogenes such as RAS and MYC can cause a reversion of tumorigenesis.
The consequences of oncogene inactivation differ depending on the type of cancer. For example, a brief inactivation of MYC oncogene was found to result in sustained regression in osteogenic sarcoma (6). By inactivating MYC, the tumor cells differentiate, and these differentiated cells cannot restore tumorigenesis by MYC reactivation. The inactivation of MYC in hepatocellular carcinoma was also found to induce sustained regression (7). However, unlike in osteogenic sarcoma, hepatocellular carcinoma is differentiated into normal liver cellular lineages, and tumorigenesis in these cells can be restored by MYC reactivation. The key point is that inactivation of the MYC oncogene can result in complete cessation of tumorigenesis, or merely induce dormancy.
There are some inhibitors of oncogenic proteins in clinical use today (Fig. 2.6.1). One such pharmacological agent is imatinib mesylate. It inactivates ABL and KIT tyrosine kinases and has been proven to be effective against chronic myelogenous leukemia and gastrointestinal stromal tumors (8). Utilizing oncogenes and their products as targets in treatment is a promising development in cancer therapeutic research.
Another general method in targeting oncogenes is through adoptive T cell therapy (ATT): using cytotoxic T cells directed against specific oncoproteins to eliminate tumors. This immunotherapy has been used for many years, and its advantage over oncogene inhibitor drugs is that the latter effect is usually transient and it selects for drug-resistant clones, resulting in tumor relapse. Several studies have shown that T cell therapy is able to eradicate large established tumors if the target antigen was cancer-driving and expressed in sufficient amounts (9). However, it is difficult to generate T cells in vitro, and the outcomes of employing ATT in the clinic have not been as ideal as expected since tumor recurrence was frequent (9). New techniques are still in development to make this potential therapy a more feasible alternative or adjuvant to inhibitor drugs.
Src as a Target for Breast Cancer Therapy
Src, the first human oncogene discovered, is a non-receptor tyrosine kinase with ubiquitous expression throughout the human body but with particularly high levels in the brain, osteoclasts and platelets (10). Under various phosphorylation conditions Src interacts with a variety of proteins in a multitude of pathways, including: cell proliferation, survival, mobility, and angiogenesis. In the case of breast cancer tumors, a common dysregulation of Src causes an increased activity of the kinase in response to elevated levels of upstream regulatory elements or signaling molecules (10), e.g. EGFR and human epidermal growth factor tyrosine kinase 2. The increase of upstream proteins forces Src to cooperate with the oncogene to increase cellular proliferation. One of the main interacting elements with Src is EGFR, which is overexpressed in 60% of breast cancers and utilizes Src to activate many of the downstream pathways mentioned above (10). Therefore, inhibition of Src kinase activity or prevention of Src-EGFR interaction may have therapeutic implications for clinical breast cancer therapy. These therapies can be grouped into three broad categories: small-molecule inhibitors of Src kinase activity; inhibitors of protein-protein interactions; and Src destabilizing agents (10). Small-molecule inhibitors to Src function by binding to Src and inhibiting its ability to transfer phosphates and therefore, directly downregulate the kinase activity of the oncoprotein. The second class of Src targeted therapies include non-peptides that inhibit Src's ability to interact with downstream substrates by sterically blocking the Src domains associated with substrate binding (10). A final class of agents used are those that destabilize Src by interfering with the molecular chaperone Hsp90. Hsp90 supports proper folding, Src stability, and intracellular localization, where interferance with this protein's activity will downregulate Src. Overall, Src inhibitors show some encouraging anti-cancer effects in vitro and in preclinical models, more work is still required before these therapeutics can be translated into practical usage for clinical breast cancer treatment (10).
BCR-ABL as a Target for CML Therapy
The Philidelphia chromosome (previously discussed) is found in over 90% of CML patients and has been shown to drive the malignancy (15). The mutation drives grow and transformation through the constitutively active protein tyrosine kinase BCR-ABL which phosphorylates other proteins in the signal transduction pathway (15). As BCR-ABL is a driving factor in the development of CML it is an ideal target for the disease. Imatinib (STI571, Gleevec), a tyrosine kinase inhibitor, was developed to combat this activity through competition with ATP at the ATP-binding site of BCR-ABL (15). This inhibits tyrosine phosphorylation of proteins interacting with BCR-ABL, quenching its constitutive activity (15). The drug has demonstrated positive outcomes in chronic phase CML patients; these patients have a ten year survival rate of 68% and an event free survival rate of 51% (16). Imatinib is also effective in treating other forms of cancer by targeting closely related tyrosine kinases (15).
Despite the efficacy of drugs that inhibit oncogenes or their protein products, tumor cells can still develop acquired resistance (AR) to the therapeutic agent. For example, EGFR tyrosine kinase inhibitors, EGFR TKI, such as geftinib and erlotinib, have been used in the treatment of lung cancer for patients with mutations in EGFR (11). The EGFR TKIs are initially successful but AR eventually occurs in all patients (11). AR occurs through secondary EGFR mutations, activation of parallel or downstream signalling pathways to bypass the inhibited pathway and phenotypic transformation (11). Consequently, therapeutic agents and strategies are being developed to combat AR (11).
One of the hypotheses behind designing new cancer therapeutics is that of oncogene addiction. This refers to a theory wherein cancer cells that express a myriad of oncogenes would be more dependent and thus "addicted" to the activity of particular oncogenes, as well as being hypersensitive to the inhibitory activity of particular tumour-suppressor genes. It has therefore been hypothesised that therapeutically pin-pointing such oncogene(s) could re-enable cancer cells to acquire a normal phenotype (12). Oncogene addiction is hypothesised to occur as a result of three currently established mechanistic models; genetic streamlining, oncogenic shock, and synthetic lethality.
Genetic streamlining refers to cancer cells continuously undergoing genetic drift, during which particular "non-essential" cellular mechanistic pathways are actively silenced. This phenomenon is described as ‘genome degradation’ whereby mutations and epigenetic modifications are acquired by any cellular processes that do not increase the fitness or viability of the tumor cells. Such silencing will have little effect on the tumor’s ability to grow, as long as the selective pressure exerted by the tumor environment remains constant. However, this decreases the number of active and functional signalling pathways available to cancer cells, which is thought to render them vulnerable to sudden changes in the tumor environment or to inhibition of the oncogene to which the cancer cells remain 'addicted' (13). Such a phenomenon has been demonstrated by Torti et al; inhibiting Met and EGFR in two different cell lines known to be ‘addicted’ to these oncogenes resulted in downregulation of Ras and PI3K driven intracellular signaling cascades, yet no signaling responses of other signaling pathways that are affected by Met and EGFR in wild-type cells (involving e.g. JNK, p38 and NF-kB) were detected. This was a validation of the hypothesis that some intracellular pathways are silenced and therefore unresponsive in tumor cells subject to oncogene addiction (13).
Oncogenic shock applies to oncogenes which are capable of regulating both pro-survival and pro-apoptotic pathways. However, it has been discussed that in transformed cells, pro-survival signaling driven by oncogene upregulation is dominant over pro-apoptotic signalling. Torti et al. have presented the examples of overexpressing BCL2 or PI3K overriding the pro-apoptotic signalling driven by the MYC oncogene (13), as well as RAS and RAF upregulation resulting in tumor cells entering senescence, which subsequently results in p53 and CDK inhibitors p16/21 being upregulated (13). As rapidly-dividing tumor cells are in a constant state of ‘hyperproliferation’, which can result in the accumulation of double strand breaks (DSB) in their DNA, caused by so-called ‘replication stress’. DSBs have been reported to induce the pathways implicated in DNA damage responses, which furthermore increases p53 and p21 driven signalling cascades. Whilst the dominance of such pro-survival signals over pro-apoptotic signals allows tumour survival, sudden targeted drug treatment of such oncogenes can prolong the pro-apoptotic signalling, yet diminishing pro-survival signalling very rapidly (14).
Synthetic lethality, refers to two oncogenes being in such a relation to each other that ablation of both oncogenes leads to cell death, whilst permitting cell survival in the presence of either of the two critical oncogenes (13). It has also been hypothesized that such relationship isn’t limited to genes implemented in parallel pathways converging in the same end product, but also to genes with very distinct functionality. Such relationships have been established in cancer cells harboring a mutation for the KRAS oncogene, in which viability of these cells is maintained only if functional STK33 (serine/threonine kinase) activity is also maintained. Therefore inhibition of STK33 / other ‘synthetic lethality partners’ of KRAS should result in a beneficial response in patients diagnosed with a KRAS-tumor, if the Synthetic Lethality hypothesis behind oncogene addiction holds true. Such validations still remain to be obtained from clinical patients (13).
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