Robert G. Bristow

Hypoxia and metabolism: Hypoxia, DNA repair and genetic instability

Areas of hypoxic tumour tissue are known to be resistant to treatment and are associated with a poor clinical prognosis. There are several reasons why this might be, including the capacity of hypoxia to drive genomic instability and alter DNA damage repair pathways. Significantly, current models fail to distinguish between the complexities of the hypoxic microenvironment and the biological effects of acute hypoxia exposures versus longer-term, chronic hypoxia exposures on the transcription and translation of proteins involved in genetic stability and cell survival. Acute and chronic hypoxia might lead to different biology within the tumour and this might have a direct effect on the design of new therapies for the treatment of hypoxic tumours.

Chk2 Is a Tumor Suppressor That Regulates Apoptosis in both an Ataxia Telangiectasia Mutated (ATM)-Dependent and an ATM-Independent Manner

ABSTRACT In response to ionizing radiation (IR), the tumor suppressor p53 is stabilized and promotes either cell cycle arrest or apoptosis. Chk2 activated by IR contributes to this stabilization, possibly by direct phosphorylation. Like p53, Chk2 is mutated in patients with Li-Fraumeni syndrome. Since the ataxia telangiectasia mutated (ATM) gene is required for IR-induced activation of Chk2, it has been assumed that ATM and Chk2 act in a linear pathway leading to p53 activation. To clarify the role of Chk2 in tumorigenesis, we generated gene-targeted Chk2-deficient mice. Unlike ATM−/− and p53−/− mice, Chk2−/− mice do not spontaneously develop tumors, although Chk2 does suppress 7,12-dimethylbenzanthracene-induced skin tumors. Tissues from Chk2−/− mice, including those from the thymus, central nervous system, fibroblasts, epidermis, and hair follicles, show significant defects in IR-induced apoptosis or impaired G1/S arrest. Quantitative comparison of the G1/S checkpoint, apoptosis, and expression of p53 proteins in Chk2−/− versus ATM−/− thymocytes suggested that Chk2 can regulate p53-dependent apoptosis in an ATM-independent manner. IR-induced apoptosis was restored in Chk2−/− thymocytes by reintroduction of the wild-type Chk2 gene but not by a Chk2 gene in which the sites phosphorylated by ATM and ataxia telangiectasia and rad3 + related (ATR) were mutated to alanine. ATR may thus selectively contribute to p53-mediated apoptosis. These data indicate that distinct pathways regulate the activation of p53 leading to cell cycle arrest or apoptosis.

Gold Nanoparticles as Radiation Sensitizers in Cancer Therapy

Abstract Among other nanoparticle systems, gold nanoparticles have been explored as radiosensitizers. While most of the research in this area has focused on either gold nanoparticles with diameters of less than 2 nm or particles with micrometer dimensions, it has been shown that nanoparticles 50 nm in diameter have the highest cellular uptake. We present the results of in vitro studies that focus on the radiosensitization properties of nanoparticles in the size range from 14–74 nm. Radiosensitization was dependent on the number of gold nanoparticles internalized within the cells. Gold nanoparticles 50-nm in diameter showed the highest radiosensitization enhancement factor (REF) (1.43 at 220 kVp) compared to gold nanoparticles of 14 and 74 nm (1.20 and 1.26, respectively). Using 50-nm gold nanoparticles, the REF for lower- (105 kVp) and higher- (6 MVp) energy photons was 1.66 and 1.17, respectively. DNA double-strand breaks were quantified using radiation-induced foci of γ-H2AX and 53BP1, and a modest increase in the number of foci per nucleus was observed in irradiated cell populations with internalized gold nanoparticles. The outcome of this research will enable the optimization of gold nanoparticle-based sensitizers for use in therapy.

DNA double-strand break repair pathway choice is directed by distinct MRE11 nuclease activities.

MRE11 within the MRE11-RAD50-NBS1 (MRN) complex acts in DNA double-strand break repair (DSBR), detection, and signaling; yet, how its endo- and exonuclease activities regulate DSBR by nonhomologous end-joining (NHEJ) versus homologous recombination (HR) remains enigmatic. Here, we employed structure-based design with a focused chemical library to discover specific MRE11 endo- or exonuclease inhibitors. With these inhibitors, we examined repair pathway choice at DSBs generated in G2 following radiation exposure. While nuclease inhibition impairs radiation-induced replication protein A (RPA) chromatin binding, suggesting diminished resection, the inhibitors surprisingly direct different repair outcomes. Endonuclease inhibition promotes NHEJ in lieu of HR, while exonuclease inhibition confers a repair defect. Collectively, the results describe nuclease-specific MRE11 inhibitors, define distinct nuclease roles in DSB repair, and support a mechanism whereby MRE11 endonuclease initiates resection, thereby licensing HR followed by MRE11 exonuclease and EXO1/BLM bidirectional resection toward and away from the DNA end, which commits to HR.

Chronic Hypoxia Decreases Synthesis of Homologous Recombination Proteins to Offset Chemoresistance and Radioresistance

Hypoxic and/or anoxic tumor cells can have increased rates of mutagenesis and altered DNA repair protein expression. Yet very little is known regarding the functional consequences of any hypoxia-induced changes in the expression of proteins involved in DNA double-strand break repair. We have developed a unique hypoxic model system using H1299 cells expressing an integrated direct repeat green fluorescent protein (DR-GFP) homologous recombination (HR) reporter system to study HR under prolonged chronic hypoxia (up to 72 h under 0.2% O(2)) without bias from altered proliferation, cell cycle checkpoint activation, or severe cell toxicity. We observed decreased expression of HR proteins due to a novel mechanism involving decreased HR protein synthesis. Error-free HR was suppressed 3-fold under 0.2% O(2) as measured by the DR-GFP reporter system. This decrease in functional HR resulted in increased sensitivity to the DNA cross-linking agents mitomycin C and cisplatin but not to the microtubule-interfering agent, paclitaxel. Chronically hypoxic H1299 cells that had decreased functional HR were relatively radiosensitive [oxygen enhancement ratio (OER), 1.37] when compared with acutely hypoxic or anoxic cells (OER, 1.96-2.61). Using CAPAN1 cells isogenic for BRCA2 and siRNA to RAD51, we confirmed that the hypoxia-induced radiosensitivity was due to decreased HR capacity. Persistent down-regulation of HR function by the tumor microenvironment could result in low-fidelity DNA repair and have significant implications for response to therapy and genetic instability in human cancers.

Hypoxia-Induced Down-regulation of BRCA1 Expression by E2Fs

Decreased BRCA1 expression in the absence of genetic mutation is observed frequently in sporadic cancers of the breast and other sites, although little is known regarding the mechanisms by which the expression of this gene can be repressed. Here, we show that activating and repressive E2Fs simultaneously bind the BRCA1 promoter at two adjacent E2F sites in vivo, and that hypoxia induces a dynamic redistribution of promoter occupancy by these factors resulting in the transcriptional repression of BRCA1 expression. Functionally, we show that hypoxia is associated with impaired homologous recombination, whereas the nonhomologous end-joining (NHEJ) repair pathway is unaffected under these conditions. Repression of BRCA1 expression by hypoxia represents an intriguing mechanism of functional BRCA1 inactivation in the absence of genetic mutation. We propose that hypoxia-induced decreases in BRCA1 expression and consequent suppression of homologous recombination may lead to genetic instability by shifting the balance between the high-fidelity homologous recombination pathway and the error-prone NHEJ pathway of DNA repair. Furthermore, these findings provide a novel link between E2Fs and the transcriptional response to hypoxia and provide insight into the mechanisms by which the tumor microenvironment can contribute to genetic instability in cancer.

Analysis of the genetic phylogeny of multifocal prostate cancer identifies multiple independent clonal expansions in neoplastic and morphologically normal prostate tissue

Genome-wide DNA sequencing was used to decrypt the phylogeny of multiple samples from distinct areas of cancer and morphologically normal tissue taken from the prostates of three men. Mutations were present at high levels in morphologically normal tissue distant from the cancer, reflecting clonal expansions, and the underlying mutational processes at work in morphologically normal tissue were also at work in cancer. Our observations demonstrate the existence of ongoing abnormal mutational processes, consistent with field effects, underlying carcinogenesis. This mechanism gives rise to extensive branching evolution and cancer clone mixing, as exemplified by the coexistence of multiple cancer lineages harboring distinct ERG fusions within a single cancer nodule. Subsets of mutations were shared either by morphologically normal and malignant tissues or between different ERG lineages, indicating earlier or separate clonal cell expansions. Our observations inform on the origin of multifocal disease and have implications for prostate cancer therapy in individual cases.

T1/T2 GLOTTIC CANCER MANAGED BY EXTERNAL BEAM RADIOTHERAPY: THE INFLUENCE OF PRETREATMENT HEMOGLOBIN ON LOCAL CONTROL

Abstract Purpose: Pretreatment hemoglobin (Hb) level has been reported to be an important prognostic factor for local control and survival in various malignancies. However, in many settings, the adverse effect of a low Hb may be related to more advanced disease. The purpose of this analysis was to assess the influence of pretreatment Hb on local control in a large series of patients with a localized cancer (T1/T2 glottic cancer, AJCC 1992) treated in a standard fashion. Materials and Methods: Between January 1981 and December 1989, 735 patients (median age 63; 657 males, 78 females) with T1/T2 glottic cancer were treated with radiation therapy (RT). The standard RT prescription was 50 Gy in 20 fractions over 4 weeks (97% of patients). Factors studied for prognostic importance for local failure included pretreatment Hb, age, sex, T category, anterior commissure involvement, subglottic extension, and tumor bulk (presence of visible tumor vs. subclinical disease). Results: With a median follow-up of 6.8 years (range 0.2–14.3), 131 patients have locally relapsed for an actuarial 5-year relapse-free rate of 81.7%. The 5-year actuarial survival was 75.8%. The mean pretreatment hemoglobin level was 14.8 g/dl and was similar in all prognostic categories. On multivariate analysis, using the Cox proportional hazards model, pretreatment Hb predicted for local failure after RT. The hazard ratio (HR) for relapse was calculated for various Hb levels. For example, the HR for a Hb of 12 g/dl vs. a Hb of 15 g/dl was 1.8 (95% confidence interval 1.2–2.5). Previously established factors, including gender, T category, subglottic extension, as well as tumor bulk, were also prognostically important for local control. Conclusions: This analysis, in a large number of similarly treated patients, indicates that pretreatment Hb is an independent prognostic factor for local control in patients with T1/T2 carcinoma of the glottis treated with RT. The underlying biology of this observation needs to be explored, and using this information, it may be possible to develop strategies to improve treatment outcome.

Spatial genomic heterogeneity within localized, multifocal prostate cancer

Herein we provide a detailed molecular analysis of the spatial heterogeneity of clinically localized, multifocal prostate cancer to delineate new oncogenes or tumor suppressors. We initially determined the copy number aberration (CNA) profiles of 74 patients with index tumors of Gleason score 7. Of these, 5 patients were subjected to whole-genome sequencing using DNA quantities achievable in diagnostic biopsies, with detailed spatial sampling of 23 distinct tumor regions to assess intraprostatic heterogeneity in focal genomics. Multifocal tumors are highly heterogeneous for single-nucleotide variants (SNVs), CNAs and genomic rearrangements. We identified and validated a new recurrent amplification of MYCL, which is associated with TP53 deletion and unique profiles of DNA damage and transcriptional dysregulation. Moreover, we demonstrate divergent tumor evolution in multifocal cancer and, in some cases, tumors of independent clonal origin. These data represent the first systematic relation of intraprostatic genomic heterogeneity to predicted clinical outcome and inform the development of novel biomarkers that reflect individual prognosis.

Germline MutY Human Homologue Mutations and Colorectal Cancer : A Multisite Case-Control Study

BACKGROUND & AIMS The MutY human homologue (MYH) gene is a member of the base-excision repair pathway involved in the repair of oxidative DNA damage. The objective of this study was to determine colorectal cancer (CRC) risk associated with mutations in the MYH gene. METHODS A total of 3811 CRC cases and 2802 controls collected from a multisite CRC registry were screened for 9 germline MYH mutations; subjects with any mutation underwent screening of the entire MYH gene. Logistic regression was used to estimate age- and sex-adjusted odds ratios (AOR). Clinicopathologic and epidemiologic data were reviewed to describe the phenotype associated with MYH mutation status and assess for potential confounding and effect modification. RESULTS Twenty-seven cases and 1 control subject carried homozygous or compound heterozygous MYH mutations (AOR, 18.1; 95% confidence interval, 2.5-132.7). CRC cases with homozygous/compound heterozygous mutations were younger at diagnosis (P=.01), had a higher proportion of right-sided (P=.01), synchronous cancers (P<.01), and personal history of adenomatous polyps (P=.003). Heterozygous MYH mutations were identified in 87 CRC cases and 43 controls; carriers were at increased risk of CRC (AOR, 1.48; 95% confidence interval, 1.02-2.16). There was a higher prevalence of low-frequency microsatellite instability (MSI) in tumors from heterozygous and homozygous/compound heterozygous MYH mutation carriers (P=.02); MSI status modified the CRC risk associated with heterozygous MYH mutations (P interaction<.001). CONCLUSIONS Homozygous/compound heterozygous MYH mutations account for less than 1% of CRC cases. Heterozygous carriers are at increased risk of CRC. Further studies are needed to understand the possible interaction between the base excision repair and low-frequency MSI pathways.