Halazonetis Lab
The long-term goal of our research is to understand cancer at the molecular level and then use this knowledge to develop novel cancer therapies. This is a very ambitious goal. Yet, because it is shared by many laboratories world-wide, there is considerable progress and hope for new effective therapies in the coming decades. Our research at the molecular level of cancer may contribute to the development of these therapies.
Research Projects
1. Activation of DNA Damage Checkpoint Pathway as a Universal Difference between Cancer and Normal Tissues
There is significant heterogeneity among cancers at the molecular level. In turn, this heterogeneity means that different therapies may need to be developed for each cancer type. For example, Gleevec, an inhibitor of the abl kinase, is very effective in treating a somewhat uncommon form of leukemia, referred to as CML, in which the abl gene is mutated, but in other cancers in which the abl gene is not mutated, Gleevec is usually not effective.
It follows from this that identifying differences that can distinguish all cancers from all normal tissues would be very important, because then such differences could be exploited for development of therapies that are effective against all types of cancer. Our laboratory, in collaboration with the laboratory of Vassilis Gorgoulis (University of Athens Medical School, Athens, Greece) and, independently, the laboratory of J. Bartek (Danish Cancer Society, Copenhagen, Denmark) have identified such a difference. The difference that we identified as distinguishing cancer cells from normal cells relates to the state of activation of the DNA damage checkpoint pathway. This is a pathway that monitors the presence of DNA damage and then, if damage is detected, elicits an appropriate cell cycle response. For example, in the presence of DNA double strand breaks (DSBs), the DNA damage checkpoint pathway will inhibit cell division until the breaks are repaired. What we found is that while in the majority of normal cells the DNA damage checkpoint pathway is not active (because in normal cells the DNA is not damaged), in human precancerous and cancerous tissues, the DNA damage checkpoint pathway is constitutively activated.
We further identified DNA replication stress as the cause for constitutive activation of the DNA damage checkpoint pathway. DNA replication stress is a term that refers to problems with DNA replication. Normally, replication forks replicate the genomic DNA without stalling. However, in cancer cells the replication forks stall or even completely collapse. After fork collapse, the DNA replication machinery needs to be re-established on the DNA for DNA replication to resume.
The reason why cancer cells have DNA replication stress is not known, but is something we are actively trying to understand. Irrespective of the reason, the presence of DNA replication stress in cancer cells, but not in normal cells, provides a therapeutic opportunity. If it were possible to inhibit re-initiation of DNA replication after fork collapse, cancer cells would not be able to replicate their genome, whereas normal cells would be unaffected. Accordingly, our laboratory tries to understand the mechanisms involved in fork collapse and replication re-initiation in cancer cells.
Figure 1. Immunohistochemistry of normal bronchial epithelium, hyperplastic (Hyper), dysplastic (Dysp) and non-small cell lung carcinoma (NSCLC) lesions.
p53(WT) and p53(Mut) are wild-type and mutant p53 genotypes, respectively. Ki67 and TUNEL staining were used to calculate proliferation and apoptotic indices, respectively. g-H2AX, phosphorylated H2AX; pT-Chk2, Chk2 phosphorylated on Thr 68. The distribution of 53BP1 in cells was examined by immunofluorescence: 53BP1 is shown in green, nuclei are outlined in blue (see our publications in Nature, 2005; 2006; Science, 2008; Nat. Rev. Mol. Cell Biol., 2010; and Mol. Oncol., 2011 for more information).
2. Functional and Structural Analysis of DNA Damage Response Proteins
Elucidating protein function at the molecular level can be greatly facilitated by structural insights. For example, we described in 2004 key insights regarding the mechanism by which the DNA damage checkpoint protein 53BP1 recognizes DNA DSBs in cells. This was made possible by determining the three-dimensional structure of the DNA DSB recognition domain of 53BP1 and then using the structure to formulate testable hypotheses. What we found is that 53BP1 recognizes methylated histone residues that become accessible in response to DNA DSBs.
More recently, we obtained insights regarding how the p53 tumor suppressor protein recognizes its target sites. The gene that encodes p53 is the most frequently mutated gene in human cancer. p53 is activated by DNA damage, including the DNA DSBs present in cancer cells, and induces cell cycle arrest or cell death. Thus, inactivation of p53 by mutations is necessary for proliferation of cancer cells harboring DNA replication stress.
The p53 protein is a transcription factor that contains sequence-specific DNA binding and homo-tetramerization domains. Interestingly, the affinities of p53 for specific and non-specific DNA sites differ by only one order of magnitude, making it hard to understand how this protein recognizes its specific DNA targets in the cell among billions of other non-specific sites. We solved by X-ray crystallography the structure of a p53 polypeptide containing both the DNA binding and oligomerization domains in complex with DNA. This structure, which is the first multidomain p53 structure at atomic resolution, reveals that sequence-specific DNA binding proceeds via an induced fit mechanism involving a conformational switch in loop L1 of the p53 DNA binding domain. Analysis of loop L1 mutants demonstrated that the conformational switch allows DNA binding off-rates to be regulated independently of affinities. We propose that these results may explain the universal prevalence of conformational switching in sequence-specific DNA binding proteins and suggest that p53 and, possibly, many other transcription factors, rely more on differences in binding off-rates, than on differences in affinities, to recognize their specific DNA sites.
Figure 2.
Left: three-dimensional structure of a multidomain p53 oligomer bound to DNA. Each p53 subunit has a different color. Right panels: detail of how subunits A (top) and B (bottom) contact DNA. In subunit A, loop L1 adopts a novel conformation that is not seen in the absence of DNA. In subunit B, loop L1 adopts the same conformation as in the absence of DNA (see our publication in EMBO Journal, 2011 for more information).