PET with 62Cu-ATSM and 62Cu-PTSM is a useful imaging tool for hypoxia and perfusion in pulmonary lesions.

OBJECTIVE: Hypoxia is a characteristic of many tumors and portends a worse prognosis in lung, cervical, prostate, and rectal cancers. Unlike the others, lung cancers present a unique challenge in measuring hypoxia, with invasive biopsies and higher rates of complications. Noninvasive imaging studies detecting hypoxia using isotopes of copper-diacetyl-bis(N4-methylthiosemicarbazone) ((62)Cu-ATSM) have predicted prognosis and treatment outcomes in some small feasibility trials. These images, however, may not identify all areas of hypoxia. Hence, we hypothesize that the addition of another PET imaging agent, copper-pyruvaldehyde-bis(N4-methylthiosemicarbazone) ((62)Cu-PTSM), which can detect areas of perfusion, can augment the information obtained in (62)Cu-ATSM PET scans. SUBJECTS AND METHODS: To characterize tumors on the basis of both perfusion and hypoxia, 10 patients were studied using both (62)Cu-ATSM and (62)Cu-PTSM PET scans. In addition, proteomic arrays looking at specific proangiogenic, survival, and proinflammatory targets were assessed. RESULTS: Six of 10 patients had evaluable PET scans. Our initial experience of characterizing lung tumor hypoxia using (62)Cu-ATSM and (62)Cu-PTSM PET scans showed that visualization of areas with hypoxia normalized for perfusion is feasible. All studied tumors exhibited some hypoxia. Despite the small sample size, a positive relationship was noted between epidermal growth factor levels and (62)Cu-ATSM-detected hypoxia. CONCLUSION: This initial series of (62)Cu-ATSM and (62)Cu-PTSM PET scans shows that evaluating lung masses by visualizing hypoxia and perfusion is a feasible and novel technique to provide more information. Further investigation is warranted to assess the potential role of (62)Cu-ATSM and (62)Cu-PTSM PET techniques combined with proteomics as alternatives to invasive biopsy techniques in clinical care.

HIF-1alpha and p53: the ODD couple?

Tumor hypoxia activates hypoxia-inducible factor-1 (HIF-1) and induces the accumulation of the tumor suppressor p53. HIF-1 signaling stimulates angiogenesis and mediates cellular adaptation to hypoxia, whereas p53 promotes hypoxia-induced apoptosis. A recent article provides in vitro biophysical evidence supporting a direct interaction between p53 and the oxygen-dependent degradation domain of the HIF-1alpha subunit. The article identifies potential structural parameters required for this interaction and suggests an alternative mechanism by which p53 might impact tumor response to therapy.

Psoralen Derivatives with Enhanced Potency.

Psoralen is a furocoumarin natural product that intercalates within DNA and forms covalent adducts when activated by ultraviolet radiation. It is well known that this property contributes to psoralen's clinical efficacy in several disease contexts, which include vitiligo, psoriasis, graft-versus-host disease and cutaneous T-cell lymphoma. Given the therapeutic relevance of psoralen and its derivatives, we attempted to synthesize psoralens with even greater potency. In this study, we report a library of 73 novel psoralens, the largest collection of its kind. When screened for the ability to reduce cell proliferation, we identified two derivatives even more cytotoxic than 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT), one of the most potent psoralens identified to date. Using MALDI-TOF MS, we studied the DNA adduct formation for a subset of novel psoralens and found that in most cases enhanced DNA binding correlated well with cytotoxicity. Generally, our most potent derivatives contain positively charged substituents, which we believe increase DNA affinity and enhance psoralen intercalation. Thus, we provide a rational approach to guide efforts toward further optimizing psoralens to fully capitalize on this drug class' therapeutic potential. Finally, the structure-activity insights we have gained shed light on several opportunities to study currently underappreciated aspects of psoralen's mechanism.

Inhibition of fatty acid synthase induces endoplasmic reticulum stress in tumor cells.

Fatty acid synthase (FAS), the cellular enzyme that synthesizes palmitate, is expressed at high levels in tumor cells and is vital for their survival. Through the synthesis of palmitate, FAS primarily drives the synthesis of phospholipids in tumor cells. In this study, we tested the hypothesis that the FAS inhibitors induce endoplasmic reticulum (ER) stress in tumor cells. Treatment of tumor cells with FAS inhibitors induces robust PERK-dependent phosphorylation of the translation initiation factor eIF2alpha and concomitant inhibition of protein synthesis. PERK-deficient transformed mouse embryonic fibroblasts and HT-29 colon carcinoma cells that express a dominant negative PERK (DeltaC-PERK) are hypersensitive to FAS inhibitor-induced cell death. Pharmacologic inhibition of FAS also induces the processing of X-box binding protein-1, indicating that the IRE1 arm of the ER stress response is activated when FAS is inhibited. Induction of ER stress is further confirmed by the increased expression of the ER stress-regulated genes CHOP, ATF4, and GRP78. FAS inhibitor-induced ER stress is activated prior to the detection of caspase 3 and PARP cleavage, primary indicators of cell death, whereas orlistat-induced cell death is rescued by coincubation with the global translation inhibitor cycloheximide. Lastly, FAS inhibitors cooperate with the ER stress inducer thapsigargin to enhance tumor cell killing. These results provide the first evidence that FAS inhibitors induce ER stress and establish an important mechanistic link between FAS activity and ER function.

The PERK/eIF2alpha/ATF4 module of the UPR in hypoxia resistance and tumor growth.

Hypoxia is a dynamic feature of the tumor microenvironment that contributes to cancer progression. In order to adapt and overcome hypoxic stress, tumor cells activate survival pathways that attempt to couple metabolic processes to reduced energy availability due to oxygen deprivation. While the hypoxia-inducible factors HIF-1 and HIF-2 are critical to the cellular response to hypoxia, HIF-independent processes are known to contribute to this adaptation. Recent evidence demonstrates that hypoxia activates components of the Unfolded Protein Response (UPR), a coordinated program that regulates cellular adaptation to increased levels of unfolded proteins in the endoplasmic reticulum (ER). Here we review the evidence implicating the ER kinase PERK, its downstream target translation initiation factor eIF2alpha, and the subsequent translational upregulation of the transcription factor ATF4 in this response. Not only are cells with compromised PERK-eIF2alpha-ATF4 signaling more sensitive to hypoxic stress in vitro but they also form tumors that grow slower in vivo with smaller hypoxic areas, indicating that the PERK-eIF2alpha-ATF4 pathway confers a survival advantage for tumor cells under hypoxia. These results, together with evidence for an involvement of other UPR pathways and ER stress proteins in hypoxia tolerance and tumor maintenance, point to a central role for UPR activation in tumor progression and suggest that this response may offer an attractive target for new anti-tumor modalities.

FAS Death Receptor: A Breast Cancer Subtype-Specific Radiation Response Biomarker and Potential Therapeutic Target.

Although a standardized approach to radiotherapy has been used to treat breast cancer, regardless of subtype (e.g., luminal, basal), recent clinical data suggest that radiation response may vary significantly among subtypes. We hypothesized that this clinical variability may be due, in part, to differences in cellular radiation response. In this study, we utilized RNA samples for microarray analysis from two sources: 1. Paired pre- and postirradiation breast tumor tissue from 32 early-stage breast cancer patients treated in our unique preoperative radiation Phase I trial; and 2. Sixteen biologically diverse breast tumor cell lines exposed to 0 and 5 Gy irradiation. The transcriptome response to radiation exposure was derived by comparing gene expression in samples before and after irradiation. Genes with the highest coefficient of variation were selected for further evaluation and validated at the RNA and protein level. Gene editing and agonistic antibody treatment were performed to assess the impact of gene modulation on radiation response. Gene expression in our cohort of luminal breast cancer patients was distinctly different before and after irradiation. Further, two distinct patterns of gene expression were observed in our biologically diverse group of breast cancer cell lines pre- versus postirradiation. Cell lines that showed significant change after irradiation were largely luminal subtype, while gene expression in the basal and HER2+ cell lines was minimally impacted. The 100 genes with the most significant response to radiation in patients were identified and analyzed for differential patterns of expression in the radiation-responsive versus nonresponsive cell lines. Fourteen genes were identified as significant, including FAS, a member of the tumor necrosis factor receptor family known to play a critical role in programed cell death. Modulation of FAS in breast cancer cell lines altered radiation response phenotype and enhanced radiation sensitivity in radioresistant basal cell lines. Our findings suggest that cell-type-specific, radiation-induced FAS contributes to subtype-specific breast cancer radiation response and that activation of FAS pathways may be exploited for biologically tailored radiotherapy.

Modulation of circulating angiogenic factors and tumor biology by aerobic training in breast cancer patients receiving neoadjuvant chemotherapy.

Aerobic exercise training (AET) is an effective adjunct therapy to attenuate the adverse side-effects of adjuvant chemotherapy in women with early breast cancer. Whether AET interacts with the antitumor efficacy of chemotherapy has received scant attention. We carried out a pilot study to explore the effects of AET in combination with neoadjuvant doxorubicin-cyclophosphamide (AC+AET), relative to AC alone, on: (i) host physiology [exercise capacity (VO2 peak), brachial artery flow-mediated dilation (BA-FMD)], (ii) host-related circulating factors [circulating endothelial progenitor cells (CEP) cytokines and angiogenic factors (CAF)], and (iii) tumor phenotype [tumor blood flow ((15)O-water PET), tissue markers (hypoxia and proliferation), and gene expression] in 20 women with operable breast cancer. AET consisted of three supervised cycle ergometry sessions/week at 60% to 100% of VO2 peak, 30 to 45 min/session, for 12 weeks. There was significant time × group interactions for VO2 peak and BA-FMD, favoring the AC+AET group (P < 0.001 and P = 0.07, respectively). These changes were accompanied by significant time × group interactions in CEPs and select CAFs [placenta growth factor, interleukin (IL)-1ß, and IL-2], also favoring the AC+AET group (P < 0.05). (15)O-water positron emission tomography (PET) imaging revealed a 38% decrease in tumor blood flow in the AC+AET group. There were no differences in any tumor tissue markers (P > 0.05). Whole-genome microarray tumor analysis revealed significant differential modulation of 57 pathways (P < 0.01), including many that converge on NF-κB. Data from this exploratory study provide initial evidence that AET can modulate several host- and tumor-related pathways during standard chemotherapy. The biologic and clinical implications remain to be determined.

Exercise behavior, functional capacity, and survival in adults with malignant recurrent glioma.

PURPOSE: Identifying strong markers of prognosis are critical to optimize treatment and survival outcomes in patients with malignant recurrent glioma. We investigated the prognostic significance of exercise behavior and functional capacity in this population. PATIENTS AND METHODS: Using a prospective design, 243 patients with WHO grades 3 to 4 recurrent malignant glioma and Karnofsky performance status (KPS) ≥ 70 completed a self-administered questionnaire that assessed exercise behavior and performed a 6-minute walk test (6MWT) to assess functional capacity. Cox proportional models were used to estimate the risk of all-cause mortality according to 6MWT distance (6MWD; < 390 meters, 390-489 meters, > 489 meters) and exercise behavior (metabolic equivalent [MET] -h/wk) adjusted for KPS and other important clinical factors. RESULTS: Median follow-up was 27.43 months. During this period, 149 deaths were recorded (61% of the total sample). Exercise behavior was an independent predictor of survival (P = .0081). Median survival was 13.03 months for patients reporting < 9 MET-h/wk relative to 21.84 months for those reporting ≥ 9 MET-h/wk. Exercise behavior added incremental prognostic value beyond that provided by KPS, age, sex, grade, and number of prior progressions (P < .001). Compared with patients reporting < 9 MET-h/wk, the adjusted hazard ratio for mortality was 0.64 (95% CI, 0.46 to 0.91) for patients reporting ≥ 9 MET-h/wk. Functional capacity was not an independent predictor of prognosis. CONCLUSION: Exercise behavior is a strong independent predictor of survival that provides incremental prognostic value to KPS as well as traditional markers of prognosis in malignant recurrent glioma.

Preferential cytotoxicity of bortezomib toward hypoxic tumor cells via overactivation of endoplasmic reticulum stress pathways.

Hypoxia is a dynamic feature of the tumor microenvironment that contributes to drug resistance and cancer progression. We previously showed that components of the unfolded protein response (UPR), elicited by endoplasmic reticulum (ER) stress, are also activated by hypoxia in vitro and in vivo animal and human patient tumors. Here, we report that ER stressors, such as thapsigargin or the clinically used proteasome inhibitor bortezomib, exhibit significantly higher cytotoxicity toward hypoxic compared with normoxic tumor cells, which is accompanied by enhanced activation of UPR effectors in vitro and UPR reporter activity in vivo. Treatment of cells with the translation inhibitor cycloheximide, which relieves ER load, ameliorated this enhanced cytotoxicity, indicating that the increased cytotoxicity is ER stress-dependent. The mode of cell death was cell type-dependent, because DLD1 colorectal carcinoma cells exhibited enhanced apoptosis, whereas HeLa cervical carcinoma cells activated autophagy, blocked apoptosis, and eventually led to necrosis. Pharmacologic or genetic ablation of autophagy increased the levels of apoptosis. These results show that hypoxic tumor cells, which are generally more resistant to genotoxic agents, are hypersensitive to proteasome inhibitors and suggest that combining bortezomib with therapies that target the normoxic fraction of human tumors can lead to more effective tumor control.