How the histological structure of some lung cancers shaped almost 70 years of radiobiology

Pivotal research led by Louis Harold Gray in the 1950s suggested that oxygen plays a vital role during radiotherapy. By proving that tumours have large necrotic cores due to hypoxia and that hypoxic cells require significantly larger doses of ionising radiation to achieve the same cell kill, Thomlinson and Gray inspired the subsequent decades of research into better defining the mechanistic role of molecular oxygen at the time of radiation. Ultimately, the work pioneered by Thomlinson and Gray led to numerous elegant studies which demonstrated that tumour hypoxia predicts for poor patient outcomes. Furthermore, this subsequently resulted in investigations into markers and measurement of hypoxia, as well as modification strategies. However, despite an abundance of pre-clinical data supporting hypoxia-targeted treatments, there is limited widespread application of hypoxia-targeted therapies routinely used in clinical practice. Significant contributing factors underpinning disappointing clinical trial results include the use of model systems which are more hypoxic than human tumours and a failure to stratify patients based on levels of hypoxia. However, translating the original findings of Thomlinson and Gray remains a research priority with the potential to significantly improve patient outcomes and specifically those receiving radiotherapy

Replication stress and chromatin context link ATM activation to a role in DNA replication

ATM-mediated signaling in response to DNA damage is a barrier to tumorigenesis. Here we asked whether replication stress could also contribute to ATM signaling. We demonstrate that, in the absence of DNA damage, ATM responds to replication stress in a hypoxia-induced heterochromatin-like context. In certain hypoxic conditions, replication stress occurs in the absence of detectable DNA damage. Hypoxia also induces H3K9me3, a histone modification associated with gene repression and heterochromatin. Hypoxia-induced replication stress together with increased H3K9me3 leads to ATM activation. Importantly, ATM prevents the accumulation of DNA damage in hypoxia. Most significantly, we describe a stress-specific role for ATM in maintaining DNA replication rates in a background of increased H3K9me3. Furthermore, the ATM-mediated response to oncogene-induced replication stress is enhanced in hypoxic conditions. Together, these data indicate that hypoxia plays a critical role in the activation of the DNA damage response, therefore contributing to this barrier to tumorigenesis

Antibody-based imaging of bioreductive prodrug release in hypoxia

Regions of hypoxia occur in most tumors and predict for poor patient prognosis. Hypoxia-activated prodrugs provide an ideal strategy to target the aggressive, hypoxic fraction of a tumor while protecting the normal tissue from toxicity. A key challenge associated with the development of novel hypoxia-activated prodrugs, however, is the ability to visualize the delivery of the prodrug to hypoxic regions and determine where it has been activated. Here we report a modified version of the commonly used nitroimidazole bioreductive group that incorporates the fluoroethyl epitope of the antibody-based hypoxia imaging agent, EF5. Attachment of this group to the red fluorescent dye, DCM, enabled us to correlate release of the DCM dye with imaging of the reduced bioreductive group using the EF5 antibody. This study confirmed that the antibody was imaging reduction and fragmentation of the pro-fluorophore. We next employed the modified bioreductive group to synthesize a new prodrug of the KDAC inhibitor Panobinostat, EF5-Pano. Release of EF5-Pano in hypoxic multiple myeloma cells was imaged using the EF5 antibody, and the presence of an imaging signal correlated with apoptosis and a reduction in cell viability. Therefore, EF5-Pano is an imageable hypoxia-activated prodrug with proven cytotoxic effect in multiple myeloma, which could be utilized in future in vivo experiments

HIF-1 alpha-independent hypoxia-induced rapid PTK6 stabilization is associated with increased motility and invasion

© 2014 Landes Bioscience. PTK6/Brk is a non-receptor tyrosine kinase overexpressed in cancer. Here we demonstrate that cytosolic PTK6 is rapidly and robustly induced in response to hypoxic conditions in a HIF-1-independent manner. Furthermore, a proportion of hypoxic PTK6 subsequently re-localized to the cell membrane. We observed that the rapid stabilization of PTK6 is associated with a decrease in PTK6 ubiquitylation and we have identified c-Cbl as a putative PTK6 E3 ligase in normoxia. The consequences of hypoxia-induced PTK6 stabilization and subcellular re-localization to the plasma membrane include increased cell motility and invasion, suggesting PTK6 targeting as a therapeutic approach to reduce hypoxia-regulated metastatic potential. This could have particular significance for breast cancer patients with triple negative disease

Effects of acute versus chronic hypoxia on DNA damage responses and genomic instability.

Questions exist concerning the effects of acute versus chronic hypoxic conditions on DNA replication and genomic stability that may influence tumorigenesis. Severe hypoxia causes replication arrest independent of S-phase checkpoint, DNA damage response, or transformation status. Arrests occur during both the initiation and elongation phases of DNA replication, correlated with a rapid decrease in available deoxynucleotide triphosphates. With fluctuating oxygen tensions in tumors, arrested hypoxic cells may undergo rapid reperfusion and reoxygenation that leads to reoxygenation-induced DNA damage. In cells subjected to chronic hypoxia, we found that replicative restart was inhibited along with numerous replication factors, including MCM6 and RPA, the latter of which limits the hypoxia-induced DNA damage response. In contrast, in cells where replicative restart occurred, it was accompanied by extensive reoxygenation-induced DNA damage and compromised DNA repair. We found that cells reoxygenated after acute hypoxia underwent rapid p53-dependent apoptosis. Our findings suggest that cells lacking functional p53 are more susceptible to genomic instability and potentially tumorigenesis if they experience reoxygenation after acute exposure to hypoxia

Hypoxia-induced transcriptional stress is mediated by ROS-induced R-loops

Hypoxia is a common feature of solid tumors and is associated with poor patient prognosis, therapy resistance and metastasis. Radiobiological hypoxia (<0.1% O2) is one of the few physiologically relevant stresses that activates both the replication stress/DNA damage response and the unfolded protein response. Recently, we found that hypoxia also leads to the robust accumulation of R-loops, which led us to question here both the mechanism and consequence of hypoxia-induced R-loops. Interestingly, we found that the mechanism of R-loop accumulation in hypoxia is dependent on non-DNA damaging levels of reactive oxygen species. We show that hypoxia-induced R-loops play a critical role in the transcriptional stress response, evidenced by the repression of ribosomal RNA synthesis and the translocation of nucleolin from the nucleolus into the nucleoplasm. Upon depletion of R-loops, we observed a rescue of both rRNA transcription and nucleolin translocation in hypoxia. Mechanistically, R-loops accumulate on the rDNA in hypoxia and promote the deposition of heterochromatic H3K9me2 which leads to the inhibition of Pol I-mediated transcription of rRNA. These data highlight a novel mechanistic insight into the hypoxia-induced transcriptional stress response through the ROS–R-loop–H3K9me2 axis. Overall, this study highlights the contribution of transcriptional stress to hypoxia-mediated tumorigenesis

Apoptosis-Specific Protein (ASP) Identified in Apoptotic Xenopus Thymus Tumor Cells

A novel apoptosis-specific protein (ASP) has recently been identified in the cytoplasm of apoptotic mammalian cells. This paper investigates whether ASP is found in Xenopus thymus tumor-derived lymphoid cell lines undergoing apoptosis and also in apoptotic, nontransformed splenocytes. Cultured Xenopus tumor lymphoid cells induced to undergo, apoptosis by serum deprivation or treatment with the calcium ionophore, ionomycin, displayed altered morphology typical of apoptotic cells, as judged by flow cytometric light-scatter characteristics and by fluorescence microscopy of acridine-orange-stained cells. Flow cytometry of permeabilized cells and fluorescence microscopy of acetone-fixed cytospins revealed that apoptotic Xenopus tumor cells, especially those displaying loss or condensation of DNA, displayed increased expression of epitopes recognized by a rabbit polyclonal antibody against ASP. Flow cytometry confirmed that ASP is also expressed in splenocytes induced to apoptose by culture in ionomycin or following concanavalin A stimulation. No increased expression of ASP was seen when lymphoid tumor cells or splenocytes were induced into necrosis by overdose with the antifungal agent amphotericin B. Western blotting with antibody against ASP identified the emergence of several protein bands in cell lysates from apoptotic, but not necrotic, Xenopus tumor cells. The new and simple methodology for identifying apoptotic cells described here is likely to be of value to those studying immune system development and associated programmed cell death in Xenopus

SPINK1 as a plasma marker for tumor hypoxia and a therapeutic target for radiosensitization

Hypoxia is associated with tumor radioresistance; therefore, a predictive marker for tumor hypoxia and a rational target to overcome it have been sought to realize personalized radiotherapy. Here, we show that serine protease inhibitor Kazal type I (SPINK1) meets these 2 criteria. SPINK1 expression was induced upon hypoxia (O2 < 0.1%) at the transcription initiation level in a HIF-dependent manner, causing an increase in secreted SPINK1 levels. SPINK1 proteins were detected both within and around hypoxic regions of xenografted and clinical tumor tissues, and their plasma levels increased in response to decreased oxygen supply to xenografts. Secreted SPINK1 proteins enhanced radioresistance of cancer cells even under normoxic conditions in EGFR-dependent and nuclear factor erythroid 2–related factor 2–dependent (Nrf2-dependent) manners and accelerated tumor growth after radiotherapy. An anti-SPINK1 neutralizing antibody exhibited a radiosensitizing effect. These results suggest that SPINK1 secreted from hypoxic cells protects the surrounding and relatively oxygenated cancer cells from radiation in a paracrine manner, justifying the use of SPINK1 as a target for radiosensitization and a plasma marker for predicting tumor hypoxia

CH-01 is a hypoxia-activated prodrug that sensitizes cells to hypoxia/reoxygenation through inhibition of Chk1 and aurora A

The increased resistance of hypoxic cells to all forms of cancer therapy presents a major barrier to the successful treatment of most solid tumors. Inhibition of the essential kinase Checkpoint kinase 1 (Chk1) has been described as a promising cancer therapy for tumors with high levels of hypoxia-induced replication stress. However, as inhibition of Chk1 affects normal replication and induces DNA damage, these agents also have the potential to induce genomic instability and contribute to tumorigenesis. To overcome this problem, we have developed a bioreductive prodrug, which functions as a Chk1/Aurora A inhibitor specifically in hypoxic conditions. To achieve this activity, a key functionality on the Chk1 inhibitor (CH-01) is masked by a bioreductive group, rendering the compound inactive as a Chk1/Aurora A inhibitor. Reduction of the bioreductive group nitro moiety, under hypoxic conditions, reveals an electron-donating substituent that leads to fragmentation of the molecule, affording the active inhibitor. Most importantly, we show a significant loss of viability in cancer cell lines exposed to hypoxia in the presence of CH-01. This novel approach targets the most aggressive and therapy-resistant tumor fraction while protecting normal tissue from therapy-induced genomic instability. © 2013 American Chemical Society

Fluorogenic platinum(iv) complexes as potential predictors for the design of hypoxia-activated platinum(iv) prodrugs

Hypoxia (low-oxygen) is one of the most common characteristics of solid tumours. Exploiting tumour hypoxia to reductively activate Pt(IV) prodrugs has the potential to deliver toxic Pt(II) selectively and thus overcome the systemic toxicity issues of traditional Pt(II) therapies. However, our current understanding of the behaviour of Pt(IV) prodrugs in hypoxia is limited. Here, we evaluated and compared the aryl carbamate fluorogenic Pt(IV) complexes, CisNap and CarboNap, as well as the previously reported OxaliNap, as potential hypoxia-activated Pt(IV) (HAPt) prodrugs. Low intracellular oxygen concentrations (<0.1%) induced the greatest changes in the respective fluorescence emission channels. However, no correlation between reduction under hypoxic conditions and toxicity was observed, except in the case for CarboNap, which displayed significant hypoxia-dependent toxicity. Other aryl carbamate Pt(IV) derivatives (including non-fluorescent analogues) mirrored these observations, where carboplatin(IV) derivative CarboPhen displayed a hypoxia-selective cytotoxicity similar to that of CarboNap. These findings underscore the need to perform extensive structure activity relationship studies on the cytotoxicity of Pt(IV) complexes under normoxic and hypoxic conditions