Essentially, three models have been put forward to elucidate the mechanisms of oncogene addiction at the molecular level. All of them take into account cell-autonomous (cancer-specific) properties and are known as (i) genetic streamlining, (ii) oncogenic shock and (iii) synthetic lethality (SL). Initially speculative, these theories were each experimentally validated and can explain different but complementary facets of this phenomenon.
The genetic streamlining hypothesis stems from the well-established notion that cancer cells undergo constant genetic drift as a consequence of the selective pressure exerted by the tumourigenic process and by the tumour microenvironment. Because of this, cancer cells are thought to lose (or, better, actively dismiss) any cellular function that has proved to be non-essential for cell viability or does not provide any increase in cellular fitness (‘genome degeneration’). At the molecular level, this occurs presumably through a mutational burden of non-adaptive alterations or epigenetic modifications (‘genetic load’). When the pressure exerted by the tumour microenvironment or by tumour-autonomous features remains constant, the genetic load in non-essential genes will have little effect on cell growth dynamics (Kamb, 2003). However, the widespread silencing of subsidiary functions renders cancer cells much more susceptible to acute perturbations: sudden changes in the composition of the surrounding stroma or inhibition of one or more of the pathways still active in cancer cells lead to rapid reduction in cellular fitness and collapse (Fig 1A). Theoretically, this process may produce an opposite outcome: an initially non-adaptive mutation can coexist as a passenger alteration along with driver mutations in the genome of a cancer cell until a new selective force – for example drug exposure – unleashes its potential to increase biological fitness in that particular circumstance; this, in some instances, can foster the emergence of resistant clones (see below).
Figure 1. Models of oncogene addiction.
The ‘genetic streamlining’ theory postulates that non-essential pathways (top, light grey) are inactivated during tumour evolution, so that dominant, addictive pathways (red) are not surrogated by compensatory signals. Upon abrogation of dominant signals, there is a collapse in cellular fitness and cells experience cell-cycle arrest or apoptosis (bottom, red to yellow shading).
In the ‘oncogenic shock’ model, addictive oncoproteins (e.g. RTKs, red triangle) trigger at the same time pro-survival and pro-apoptotic signals (top, red and blue pathway, respectively). Under normal conditions, the pro-survival outputs dominate over the pro-apoptotic ones (top), but following blockade of the addictive receptor, the rapid decline in the activity of survival pathways (dashed lines, bottom) subverts this balance in favour of death-inducing signals, which tend to last longer and eventually lead to apoptotic death.
Two genes are considered to be in a synthetic lethal relationship when loss of one or the other is still compatible with survival but loss of both is fatal. In the top panel, biochemical inactivation of pathway A (grey) has no effect on cell viability because pathway B (red), which converges at some point on a common substrate or effector (yellow), has compensating activity. When the integrity of pathway B is disrupted (bottom), the common downstream biochemical function is lost and again cancer cells may experience cell cycle arrest or apoptosis.
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Inactivation of signalling pathways in cancer cells after genetic drift may also occur at the biochemical or transcriptional levels as a consequence of chronic oncogenic signalling. Indeed, the unrelenting activity of dominant oncogenes is likely to be counteracted by a certain extent of reactive adaptation, including activation of compensatory pathways and positive or negative feedback loops. Using phosphoproteomic and gene expression profiling, we have recently demonstrated the presence of ‘sensitive’ and ‘indifferent’ pathways in cell lines addicted to the MET oncogene – encoding the Met tyrosine kinase receptor for hepatocyte growth factor (HGF) – or to epidermal growth factor receptor (EGFR). Met or EGFR inhibition in these settings results in the selective decline of RAS- and PI3K-dependent cascades, whilst many other signals known to affect Met- and EGFR-driven proliferation in non-addicted cells – including JNK, p38, STATs and NF-κB – remain active or exhibit scant responses (Bertotti et al, 2009). In the context of genetic streamlining, this piece of information corroborates the notion that cancer cells host large arrays of indolent and functionally neutral pathways and small ensembles of functionally active, self-sufficient transducers. The presence of only a limited subset of operational signalling nodes and the absence of buffering circuits reveal the vulnerability of the oncogene addiction state.
Settleman and colleagues have proposed a model referred to as ‘oncogenic shock’. The fundamental premise to this concept is that most dominant oncogenes are able to sustain at the same time both pro-survival and pro-apoptotic signals (Sharma and Settleman, 2007). This duality is an in-built property of normal cells, in which strong oncogenic insults can counteract excessive pro-mitogenic signals induced by the same molecule through concomitant induction of apoptosis. In transformed cells, this intrinsic apoptotic defense is disabled for many reasons, including the fact that the pro-survival signals that emanate from hyperactive oncoproteins tend to dominate over the parallel pro-apoptotic outputs (Fig 1B). A variety of experimental data, collected in diverse cellular and in vivo transgenic models, is in favour of this notion: for example the MYC oncogene displays apoptosis-inducing properties in low-serum conditions (Evan et al, 1992), which can be inhibited by overexpression of the anti-apoptotic BCL2 protein (Bissonnette et al, 1992) or PI3K/AKT pathway activation (Kauffmann-Zeh et al, 1997). In normal physiology, the pro-apoptotic function of MYC is apparent during development, as it causes the negative selection of T lymphocytes upon antigen stimulation (Shi et al, 1992). Similarly, continued overexpression of the RAS or RAF oncogenes in primary human cells induces cell cycle arrest through activation of the MAPK family members p38 and JNK, which in turn induce transcriptional upregulation of p53 or the cyclin-dependent kinase (CDK) inhibitors p16 and/or p21 (Fanton et al, 2001; Zhu et al, 1998). As an alternative to active induction of apoptosis, some cells react to relentless oncogenic signalling by entering a state of senescence, that is an irreversible condition of post-mitotic dormancy. In vitro and in vivo models have shown that hyperproliferating cells accumulate genomic lesions such as DNA double strand breaks (DSB), a process known as ‘replication stress’. In turn, DSBs engage the DNA damage response machinery, which activates the prototypical effector ataxia telangiectasia mutated (ATM); this converges on either p53 or p21, triggering the proliferation blockade that typifies the senescent phenotype (Bartkova et al, 2006; Di Micco et al, 2006; Halazonetis et al, 2008).
The oncogenic shock hypothesis relies on the experimental observation that targeted disruption of signal-generating oncoproteins results in differential kinetics of downstream signal decay: anti-apoptotic effectors (such as ERKs, AKT and STATs) display rapid diminution of activity; conversely, death-inducing molecules (namely p38) display delayed accumulation. This temporal imbalance has been demonstrated in a variety of cellular systems driven by oncogenically active tyrosine kinases, including BCR-ABL, SRC and EGFR (Sharma et al, 2006; Sharma and Settleman, 2010b). The oncogenic shock hypothesis deserves at least two comments. First, it postulates that the apoptotic response observed following abrogation of addictive oncoproteins is an active process of signal-mediated induction of cell death; this is in contrast to the passive occurrence of signal deprivation predicted in the genetic streamlining model. Second, the ‘potency’ of the oncogenic signal in generating pro-survival and pro-apoptotic outputs seems to be more crucial than the temporal appearance of the dominant genetic lesion. While it can be intuitive to think that an initiating oncogene will be more influential as a dominant alteration than genetic lesions occurring subsequently during tumour evolution, we can also reasonably argue that addictive oncogenes with powerful pro-apoptotic activity are likely to arise late during the tumour's natural history, when at least some apoptotic safeguards have been disengaged; otherwise, cells would die, and oncogene hyperactivity would be negatively selected.
The theory of SL states that a gene A is in a synthetic lethal relationship with a gene B when loss of function of either gene A or gene B is fully compatible with cell viability, whereas, loss of activity of both A and B gene products is lethal for the cell (Kaelin, 2005). This notion is rather intuitive when genes A and B belong to alternative metabolic (enzymatic) chains with a common end-product; but, at least in principle, it can also be applied to signalling axes driving more sophisticated and integrated cellular functions, such as survival and proliferation (Fig 1C). The concept of SL dates back to the beginning of the last century and was initially applied to account for experimental data obtained in single-celled organisms, such as bacteria and yeast. In the past decade, it has been exploited for the characterization of orthologue enzymes involved in certain metabolic pathways in multi-cellular model organisms (Caenorhabditis elegans, Drosophila melanogaster) and, more recently, it has been extended to human tumour cell lines (Brough et al, 2011; Nijman, 2011). In cancer, SL is proposed to occur when alteration of a gene (e.g. its genetic silencing or pharmacologic inactivation) results in cell death only in the presence of another non-lethal genetic alteration (e.g. a cancer-associated mutation). Because the gene, that is synthetic lethal in combination with the cancerous mutation is usually in its wild-type form, SL is also defined as ‘non-oncogene addiction’.
The SL working hypothesis led to the discovery of the synthetic lethal interaction between the BRCA1 and/or BRCA2 gene products and [poly(ADP-ribose)-polymerase] PARP-1 protein (Lord and Ashworth, 2008). BRCA1 and BRCA2 have a key role in homologous recombination (HR; Fig 2A), an important pathway for DNA damage repair, and their inactivation results in defective restoration of DNA DSBs (see above). Single-strand breaks (SSBs) must also be repaired in BRCA-deficient cells, because SSBs can turn into lethal DSBs following DNA replication. The DNA repair pathway principally involved in repairing SSBs is base-excision repair (BER; Fig 2B), and one of the proteins essential for BER is PARP-1 (Rouleau et al, 2010). It has been demonstrated that, when BER is inactivated (e.g. as a consequence of PARP1 inhibition), BRCA-deficient cells are unable to repair the DSBs that evolve from SSBs, and this leads to deadly defects in the genome (Bryant et al, 2005; Farmer et al, 2005). Interestingly, acquired resistance to PARP inhibitors in cells with BRCA2 loss-of-function alterations may result from late ‘reverting’ mutations, that is new deletions in the context of the BRCA2 gene that restore the normal open reading frame abrogated by the original genetic lesion, thus rendering the cells once again capable to perform HR (Edwards et al, 2008; Sakai et al, 2008). Of note, two studies recently suggested an alternative route for restoration of HR competence in BRCA1-deleted cells (Bouwman et al, 2010; Bunting et al, 2010). Tumour protein p53 binding protein 1 (TP53BP1, also known as 53BP1) is a component of the non-homologous end joining (NHEJ) repair system (Fig 2C), which acts by binding to chromatid breaks and triggers the formation of aberrant fusions between heterologous chromosomes. These fusion products can represent ‘toxic’ rearrangements and negatively affect cell viability. Mechanistically, 53BP1 blunts the ATM-mediated resection of the broken strands, ultimately impeding HR (Bunting et al, 2010). HR is the preferred repair type for DSBs in wild-type cells, whereas, in BRCA1-deficient cells (in which normal HR is compromised) the balance switches towards the NHEJ system. Suppression of residual HR function, for example through PARP inhibition, further enhances the ‘toxic’ switch to NHEJ, suggesting that neutralization of NHEJ might alleviate generalized DNA damage and counteract the effects of PARP inhibition. Indeed, RNAi-mediated depletion of 53BP1 reduces hypersensitivity of BRCA1 mutant cells to PARP inhibitors and leads to partial restoration of HR: in genetic terminology, 53BP1 loss is said to be ‘synthetic viable’ with BRCA1 deficiency (Aly and Ganesan, 2011; Kaelin, 2005). The clinical significance of these findings is still unclear: BRCA1-associated cancers often exhibit reduced expression of 53BP1, which might have predictive value as a potential biomarker of therapeutic resistance to PARP inhibitors (Bouwman et al, 2010).
Figure 2. Modes of DNA damage repair.
HR safeguards genome integrity in late S/G2 phases of the cell cycle and relies on the ability to use the recently formed sister chromatids as a template to guide repair of the damaged strands. Exonuclease activity produces two single-stranded (ss) DNA ends, one at each side of a double-strand (ds) DNA break (top). Each of the two ssDNA ends pair with complementary sequences of the sister chromatid (unwinding of sister chromatids is not represented here for simplicity); elongation is then performed by DNA polymerase. Subsequent release from the sister chromatid, pairing of the elongated ssDNA ends, further elongation and, ultimately, ligation give rise to the wild-type sequence. BRCA1 and BRCA2, among many other proteins, are part of the HR machinery.
BER senses chemically altered bases (β) with a minimal effect on double helix topology. Glycosylases first cleave the bond linking the base with the deoxyribose (middle top); this is in turn excised by an apurinic/apyrimidinic endonuclease (APE) with high affinity for base-free sugars (middle bottom). DNA polymerase β replaces the missing nucleotide, which is finally ligated to reconstitute the correct sequence.
When homologous sequences are not available as templates, the ssDNA ends generated by exonucleases can be joined together by a small number of base pairs. This event if followed by filling of the gaps in each strand and ligation of any remaining ssDNA breaks. Of note, the sequence repaired by this NHEJ repair system lacks some of the bases originally present in the undamaged DNA and is therefore intrinsically error-prone.
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In addition to applying the SL concept to tumour suppressor genes, an analogous strategy has been successfully implemented for dominantly acting oncogenes and specifically for KRAS. Considering that KRAS is a widespread target in human malignancies that remains substantially undruggable, the inhibition of its putative synthetic lethal partners was considered an affordable way to indirectly abrogate KRAS pro-tumourigenic activity. Using diverse screening approaches, several groups identified many putative SL interactors of KRAS. Singh et al used RNAi to deplete the KRAS protein in lung and pancreatic cancer cell lines harbouring mutated KRAS alleles; this identified KRAS-dependent cells (which reacted to KRAS silencing with growth impairment) and KRAS-independent cells (whose proliferative activity was not affected by KRAS silencing, despite the presence of activating KRAS mutations). The KRAS-dependent cells were characterized by the expression of proteins – including the tyrosine kinases SYK, RON, and integrin β6 – that resulted in a differentiated epithelial phenotype and that, when inactivated, induced epithelial–mesenchymal transition and apoptosis (Singh et al, 2009).
A high-throughput, RNAi-mediated SL screen in KRAS-mutated cancer cells of different tissues revealed that the serine/threonine kinase STK33 is selectively required in these cells to sustain viability. This is mechanistically supported by the observation that STK33 suppresses mitochondrial apoptosis via S6K1-mediated inactivation of the pro-apoptotic effector BAD, although the signalling intermediates between STK33 and S6K1 are still to be characterized (Scholl et al, 2009). With a similar approach, Barbie et al discovered the anti-apoptotic role of the non-canonical IκB kinase TBK1, an upstream activator of NF-κB signalling, in KRAS-addicted cancer cell lines; this introduced NF-κB-related signals as a ‘co-dependent’ survival pathway in KRAS-mutated cancers (Barbie et al, 2009). An independent genome-wide RNAi screen in isogenic cell lines differing only in the presence or absence of a KRAS mutation identified 77 SL candidate genes. Computational analysis supported an increased dependency of mutated cells on proteins involved in the mitotic machinery (e.g. polo-like kinase 1, PLK1) and the proteasome suggesting that KRAS mutation is associated with mitotic stress; intriguingly, gene expression analysis revealed a correlation between decreased expression of key mitotic proteins and longer survival of patients bearing tumours featuring a RAS transcriptional signature (Luo et al, 2009). Finally, the effects of depletion of different CDKs were studied in cells derived from mice engineered to endogenously express a mutant KRAS allele and in cell lines derived from KRAS mutated, non-small cell lung carcinomas (NSCLCs). Selective loss of CDK4 had the most prominent negative effect on the cancerous phenotype by inducing a strong senescence response (Puyol et al, 2010).
The ultimate validation of all these SL strategies will be evidence that patients with KRAS-mutated tumours clinically benefit from treatment with inhibitors of the identified SL partners. Selective CDK4 inhibitors are available for use in humans but so far have shown only modest therapeutic efficacy in unselected populations of patients with leukaemia or breast tumours. Based on the novel results discussed here, such inhibitors warrant future investigation in the context of KRAS-mutated NSCLCs (Krystof and Uldrijan, 2010; Shapiro, 2006). Similarly, PLK1 inhibitors are in the early phases of clinical development and, likely, will soon be tested in patients with KRAS-mutated cancers (Strebhardt, 2010).
Lastly, in addition to models that rely on cell-autonomous properties, recent data from Felsher and colleagues suggest that non-cell autonomous effects might deserve consideration in the context of oncogene addiction and ‘synthetic lethal’ heterologous cell–cell interactions. In particular, functional CD4+ T-lymphocytes appear to be needed for efficient induction of cellular senescence and/or inhibition of angiogenesis upon inactivation of the MYC or BCR-ABL oncogenes in mouse models of T cell acute lymphoblastic lymphoma and pro-B cell leukaemia, respectively (Rakhra et al, 2010).