DNA damage response: A barrier or a path to tumor progression?

Why do some tumors grow and spread rapidly, while others remain localized or grow slowly? Perhaps some tumors never make it past one transformed cell? Recent advances in genomics, proteomics, and other “-omics” have provided a plethora of data on molecular aberrations in malignant cells, but in most cases these data are hard to make sense of and so are currently clustered as “molecular signatures”. Through clinical experience, these “signatures” can be associated with the aggressiveness of the malignant disease and serve as guidelines for prognosis and selection of the form of treatment of the disease, but progress is likely to be slow until new concepts are generated from these data. Thus, understanding of the pathways that signal biological outcomes appears a critical priority. The report by Oka et al. in this issue of Cancer Biology & Therapy on DNA damage signaling draws attention to one such pathway, also known as the DNA Damage Response (DDR).¹

In normal mammalian cells, DNA damage activates DNA repair genes, and this is signaled by ATM (Ataxia Telangiectasia Mutated), a kinase that activates another kinase, Chk2 (check point kinase 2), which can promote intra-S and G₂ phase cell cycle arrest, as well as apoptosis. A parallel pathway consisting of ATR, an ATM related kinase, and Chk1 has similar outcomes, though there are some differences, as ATR is reported to have an essential role during DNA replication, and senses its blockage.^2-5 These pathways converge on the p53 tumor suppressor gene “the guardian of the genome”,⁶ which can activate molecules such as p21^Waf1,⁷ that arrest cell cycle progression to allow DNA damage to be repaired (Fig. 1).⁸ Alternatively, if the situation seems hopeless, p53 nudges the cell to self-destruction by enhancing the expression of pro-apoptotic molecules such as Bax or Bim.^9,10 In either case, the cells do not self-renew so the mutations, some of which may cause self-generation of growth signals or survival under adverse conditions, are “nipped in the bud”. Thus, the DNA damage signaling proteins interact upstream with DNA repair proteins, and downstream with regulators of cell cycle progression and cell survival. The upstream components include those that promote increased DNA accessibility in the chromatin and localization of DNA repair complexes. A specific example is the phosphorylation by ATM/ATR of a variant of nucleosome core histone H2A known as H2AX, and its phosphorylated form (γH2AX) is a useful marker of DDR,⁵ as it is recognized by a specific antibody in tissue sections as discrete nuclear foci, where DNA repair is presumed to take place.

Figure 1.

DNA Damage Response (DDR) pathways in normal and malignant cells. In normal cells, different types of genotoxic stress (e.g., DNA double-strand breaks) or DNA replication stress elicit DDR as part of the self-defense system. This involves the activation (via phosphorylation) of “sensor” kinases, ATM and ATR, which in turn activate Chk2 and Chk1 checkpoint kinases, respectively, all leading to phosphorylation of histone H2AX at the sites of DNA damage. These events invoke multiple downstream pathways which, depending on the extent of DNA damage, induce either cell cycle arrest (permitting DNA repair) or apoptosis (in case of irreparable DNA damage). Thus, cell cycle arrest can result from Chk1/2-dependent activating phosphorylation of p53 tumor suppressor gene or inactivating phosphorylation of Cdc25 family phosphatases which lead, respectively, to an upregulation of cyclin-dependent kinase inhibitor p21^Waf1, or inhibition of CDK2 activity by rendering it in an inactive (phosphorylated) state. Alternatively, Chk1/2-dependent activation of p53 can induce apoptosis via upregulation and activation of a subset of pro-apoptotic proteins. A similar apoptotic effect may result from ATM-dependent phosphorylation of the nonreceptor tyrosine kinase c-Abl,²⁴ which phosphorylates and activates the p53-related protein p73,²⁵,²⁶ leading to induction of pro-apoptotic genes in a manner that overlaps with the effect of p53 (reviewed in ref. 27). Interestingly, besides promoting cell cycle arrest, DDR-triggered inhibition of CDK2 may also contribute to the induction of pro-apoptotic genes by rendering the transcription factor FOXO-1 in its active (unphosphorylated) state.²¹ In accordance with the data obtained by Oka et al.¹ colon tumorigenesis is associated with a gradual activation of the upstream DDR elements (ATM, Chk2 and H2AX) but, paradoxically, this does not result in an increased apoptosis induction, suggesting that malignant transformation may result in impairments of the DDR pathways downstream from Chk1/2 and the ATM-cAbl pathway (indicated by red crosses).

In the early stages of tumorigenesis, one molecular lesion appears to be sufficient to alter the growth properties of the transformed cell. For instance, colorectal carcinogenesis may start as an inherited, or more commonly environmentally acquired, mutation of the APC (Adenomatous Polyposis Coli) gene, which propels excessive, but not invasive, growth of the cells in colonic mucosa.¹¹ But this single molecular lesion predisposes a cell to further sequential accumulation of other mutations, including those of p53, and the cells acquire invasive properties (Fig. 2). This is known as genomic instability, and is generally due to the faulty repair of DNA lesions, which may be incidental to the replicative process itself or due to mutagens in the environment.¹² While normal cells are not subject to this hazard, as they have functioning cell cycle checkpoints that prevent cells with DNA damage from entering S phase, as recognized back in 1973;¹³ transformed cells are at risk for further damage, since these check points are usually not functional.^14-16

Figure 2.

Possible role of DDR impairment in tumorigenesis. DDR protects normal cells from consequences of DNA damage through the activation of cell cycle checkpoints which prevent entry into the S phase of cells with damaged DNA. In the early stages of tumorigenesis, initiating mutations and associated DNA replication stress cause a marked activation of DDR. This frequently provides a sufficient barrier to the progression of precancerous lesions into malignant tumors by inducing cell cycle arrest and eliminating malignant cells by apoptosis induction. However, genomic instability and accumulating mutations can still lead to a more aggressive cancer, in spite of further activation of the upstream DDR components, as shown by Oka et al. in this issue of CB&T.¹ This loss of the protecting function of the DDR may be explained by the accumulating defects in the downstream apoptosis-inducing and cell cycle-inhibitory pathways that are normally activated by DNA damage.

Yet in spite of this grim scenario, not everyone develops aggressive tumors. This is known to be due, in part, to elimination of transformed cells by the immune system, which recognizes new cell surface components. It has, however, also been proposed by groups led by Bartek¹⁷ and by Halazonetis¹⁸ that additional defenses against tumor progression are provided by DDR. These groups have provided evidence, based partly on studies of excised human bladder tumors at various stages of neoplastic progression,¹⁹ and precancerous lesions of lung and skin,²⁰ that DDR may provide a barrier to neoplastic progression in early tumorigenesis. This seems reasonable, because sensors such as ATM/ATR can detect DNA damage and alert the DNA repair machinery, as well as Chk1/Chk2 and p53 or the related protein p73, to induce cell cycle arrest or apoptosis, and thus provide selective pressure against early neoplastic progression. However, the study by Oka et al. reported in this issue of Cancer Biology & Therapy¹ raises the possibility that DDR may, under some circumstances, actually enhance the likelihood of malignant transformation.

Oka et al. observed the presence of activated markers of DDR in tumors from 55 consecutive cases of colorectal carcinoma. The markers, p-ATM, γH2AX and p-Chk2 protein levels, were found to be present in minimal amounts in normal tissue adjacent to the tumors, but their level increased in parallel with the progression from adenoma to advanced carcinoma. Intriguingly, no apoptosis, indicative of major DNA damage, was detected. The authors suggest that this is due to a malfunction of the apoptotic cascade or to an impairment of the downstream components of DDR. These were not identified, but the authors speculate that the impairment of DDR was due to the inability of the transcription factor FOXO-1 to induce apoptosis of cells with severe DNA damage. Normally, Chk2-dependent Cdk2 inactivation during DDR renders FOXO-1 in an unphosphorylated “pro-apoptotic” state;²¹ however, this as well as other downstream DDR pathways might be disabled in advanced cancers (Figs. 1 and 2). Thus, DDR may prevent, but under different conditions promote, tumor progression by allowing cells with increasingly severe DNA damage to survive under some circumstances.

Of course, this concept requires further documentation and an exploration of the possible ways of targeting DDR for treatment and/or chemoprevention of cancer. Therapeutically relevant DDR inhibition approaches have been described by Ljungman in a recent review.²² On the other hand, facilitation of DDR, as a cellular defense system, may have a role in cancer prevention. Indeed, recent findings suggest that frequently consumed plant antioxidants may cause DNA damage²³ and therefore their regular ingestion may reduce cancer incidence, acting, at least in part, by DDR activation. Thus, studies by Oka et al. may have clinical and preventive importance by expanding our knowledge of the DDR.