Metabolic fingerprinting by nuclear magnetic resonance of hepatocellular carcinoma cells during p53 reactivation-induced senescence.

Cellular senescence is characterized by stable cell cycle arrest. Senescent cells exhibit a senescence-associated secretory phenotype that can promote tumor progression. The aim of our study was to identify specific nuclear magnetic resonance (NMR) spectroscopy-based markers of cancer cell senescence. For metabolic studies, we employed murine liver carcinoma Harvey Rat Sarcoma Virus (H-Ras) cells, in which reactivation of p53 expression induces senescence. Senescent and nonsenescent cell extracts were subjected to high-resolution proton (1H)-NMR spectroscopy-based metabolomics, and dynamic metabolic changes during senescence were analyzed using a magnetic resonance spectroscopy (MRS)-compatible cell perfusion system. Additionally, the ability of intact senescent cells to degrade the extracellular matrix (ECM) was quantified in the cell perfusion system. Analysis of senescent H-Ras cell extracts revealed elevated sn-glycero-3-phosphocholine, myoinositol, taurine, and creatine levels, with decreases in glycine, o-phosphocholine, threonine, and valine. These metabolic findings were accompanied by a greater degradation index of the ECM in senescent H-Ras cells than in control H-Ras cells. MRS studies with the cell perfusion system revealed elevated creatine levels in senescent cells on Day 4, confirming the 1H-NMR results. These senescence-associated changes in metabolism and ECM degradation strongly impact growth and redox metabolism and reveal potential MRS signals for detecting senescent cancer cells in vivo.

Acidosis-mediated increase in IFN-γ-induced PD-L1 expression on cancer cells as an immune escape mechanism in solid tumors.

Immune checkpoint inhibitors have revolutionized cancer therapy, yet the efficacy of these treatments is often limited by the heterogeneous and hypoxic tumor microenvironment (TME) of solid tumors. In the TME, programmed death-ligand 1 (PD-L1) expression on cancer cells is mainly regulated by Interferon-gamma (IFN-γ), which induces T cell exhaustion and enables tumor immune evasion. In this study, we demonstrate that acidosis, a common characteristic of solid tumors, significantly increases IFN-γ-induced PD-L1 expression on aggressive cancer cells, thus promoting immune escape. Using preclinical models, we found that acidosis enhances the genomic expression and phosphorylation of signal transducer and activator of transcription 1 (STAT1), and the translation of STAT1 mRNA by eukaryotic initiation factor 4F (elF4F), resulting in an increased PD-L1 expression. We observed this effect in murine and human anti-PD-L1-responsive tumor cell lines, but not in anti-PD-L1-nonresponsive tumor cell lines. In vivo studies fully validated our in vitro findings and revealed that neutralizing the acidic extracellular tumor pH by sodium bicarbonate treatment suppresses IFN-γ-induced PD-L1 expression and promotes immune cell infiltration in responsive tumors and thus reduces tumor growth. However, this effect was not observed in anti-PD-L1-nonresponsive tumors. In vivo experiments in tumor-bearing IFN-γ-/- mice validated the dependency on immune cell-derived IFN-γ for acidosis-mediated cancer cell PD-L1 induction and tumor immune escape. Thus, acidosis and IFN-γ-induced elevation of PD-L1 expression on cancer cells represent a previously unknown immune escape mechanism that may serve as a novel biomarker for anti-PD-L1/PD-1 treatment response. These findings have important implications for the development of new strategies to enhance the efficacy of immunotherapy in cancer patients.

Evaluating near-infrared photoimmunotherapy for targeting fibroblast activation protein-α expressing cells in vitro and in vivo.

Photoimmunotherapy (PIT), carried out using an Ab conjugated to the near infrared dye IRDye700DX, is achieving significant success in target-specific elimination of cells. Fibroblast activation protein alpha (FAP-α) is an important target in cancer because of its expression by cancer-associated fibroblasts (CAFs) as well as by some cancer cells. Cancer-associated fibroblasts that express FAP-α have protumorigenic and immune suppressive functions. Using immunohistochemistry of human breast cancer tissue microarrays, we identified an increase of FAP-α+  CAFs in invasive breast cancer tissue compared to adjacent normal tissue. We found FAP-α expression increased in fibroblasts cocultured with cancer cells. In proof-of-principle studies, we engineered human FAP-α overexpressing MDA-MB-231 and HT-1080 cancer cells and murine FAP-α overexpressing NIH-3T3 fibroblasts to evaluate several anti-FAP-α Abs and selected AF3715 based on its high binding affinity with both human and mouse FAP-α. After conjugation of AF3715 with the phthalocyanine dye IR700, the resultant Ab conjugate, FAP-α-IR700, was evaluated in cells and tumors for its specificity and effectiveness in eliminating FAP-α expressing cell populations with PIT. Fibroblast activation protein-α-IR700-PIT resulted in effective FAP-α-specific cell killing in the engineered cancer cells and in two patient-derived CAFs in a dose-dependent manner. Following an intravenous injection, FAP-α-IR700 retention was three-fold higher than IgG-IR700 in FAP-α overexpressing tumors, and two-fold higher compared to WT tumors. Fibroblast activation protein-α-IR700-PIT resulted in significant growth inhibition of tumors derived from FAP-α overexpressing human cancer cells. A reduction of endogenous FAP-α+ murine CAFs was identified at 7 days after FAP-α-IR700-PIT. Fibroblast activation protein-α-targeted near infrared PIT presents a promising strategy to eliminate FAP-α+ CAFs.

Cancer insights from magnetic resonance spectroscopy of cells and excised tumors.

Multinuclear ex vivo magnetic resonance spectroscopy (MRS) of cancer cells, xenografts, human cancer tissue, and biofluids is a rapidly expanding field that is providing unique insights into cancer. Starting from the 1970s, the field has continued to evolve as a stand-alone technology or as a complement to in vivo MRS to characterize the metabolome of cancer cells, cancer-associated stromal cells, immune cells, tumors, biofluids and, more recently, changes in the metabolome of organs induced by cancers. Here, we review some of the insights into cancer obtained with ex vivo MRS and provide a perspective of future directions. Ex vivo MRS of cells and tumors provides opportunities to understand the role of metabolism in cancer immune surveillance and immunotherapy. With advances in computational capabilities, the integration of artificial intelligence to identify differences in multinuclear spectral patterns, especially in easily accessible biofluids, is providing exciting advances in detection and monitoring response to treatment. Metabolotheranostics to target cancers and to normalize metabolic changes in organs induced by cancers to prevent cancer-induced morbidity are other areas of future development.

Twist alters the breast tumor microenvironment via choline kinase to facilitate an aggressive phenotype.

Twist (TWIST1) is a gene required for cell fate specification in embryos and its expression in mammary epithelium can initiate tumorigenesis through the epithelial-mesenchymal transition. To identify downstream target genes of Twist in breast cancer, we performed microarray analysis on the transgenic breast cancer cell line, MCF-7/Twist. One of the targets identified was choline kinase whose upregulation resulted in increased cellular phosphocholine and total choline containing compounds-a characteristic observed in highly aggressive metastatic cancers. To study the interactions between Twist, choline kinase, and their effect on the microenvironment, we used 1H magnetic resonance spectroscopy and found significantly higher phosphocholine and total choline, as well as increased phosphocholine/glycerophosphocholine ratio in MCF-7/Twist cells. We also observed significant increases in extracellular glucose, lactate, and [H +] ion concentrations in the MCF-7/Twist cells. Magnetic resonance imaging of MCF-7/Twist orthotopic breast tumors showed a significant increase in vascular volume and permeability surface area product compared to control tumors. In addition, by reverse transcription-quantitative polymerase chain reaction, we discovered that Twist upregulated choline kinase expression in estrogen receptor negative breast cancer cell lines through FOXA1 downregulation. Moreover, using The Cancer Genome Atlas database, we observed a significant inverse relationship between FOXA1 and choline kinase expression and propose that it could act as a modulator of the Twist/choline kinase axis. The data presented indicate that Twist is a driver of choline kinase expression in breast cancer cells via FOXA1 resulting in the generation of an aggressive breast cancer phenotype.

Deep learning-based classification of preclinical breast cancer tumor models using chemical exchange saturation transfer magnetic resonance imaging.

Chemical exchange saturation transfer (CEST) magnetic resonance imaging has shown promise for classifying tumors based on their aggressiveness, but CEST contrast is complicated by multiple signal sources and thus prolonged acquisition times are often required to extract the signal of interest. We investigated whether deep learning could help identify pertinent Z-spectral features for distinguishing tumor aggressiveness as well as the possibility of acquiring only the pertinent spectral regions for more efficient CEST acquisition. Human breast cancer cells, MDA-MB-231 and MCF-7, were used to establish bi-lateral tumor xenografts in mice to represent higher and lower aggressive tumors, respectively. A convolutional neural network (CNN)-based classification model, trained on simulated data, utilized Z-spectral features as input to predict labels of different tissue types, including MDA-MB-231, MCF-7, and muscle tissue. Saliency maps reported the influence of Z-spectral regions on classifying tissue types. The model was robust to noise with an accuracy of more than 91.5% for low and moderate noise levels in simulated testing data (SD of noise less than 2.0%). For in vivo CEST data acquired with a saturation pulse amplitude of 2.0 µT, the model had a superior ability to delineate tissue types compared with Lorentzian difference (LD) and magnetization transfer ratio asymmetry (MTRasym ) analysis, classifying tissues to the correct types with a mean accuracy of 85.7%, sensitivity of 81.1%, and specificity of 94.0%. The model's performance did not improve substantially when using data acquired at multiple saturation pulse amplitudes or when adding LD or MTRasym spectral features, and did not change when using saliency map-based partial or downsampled Z-spectra. This study demonstrates the potential of CNN-based classification to distinguish between different tumor types and muscle tissue, and speed up CEST acquisition protocols.

Transport-driven engineering of liposomes for delivery of α-particle radiotherapy to solid tumors: effect on inhibition of tumor progression and onset delay of spontaneous metastases.

PURPOSE: Highly cytotoxic α-particle radiotherapy delivered by tumor-selective nanocarriers is evaluated on metastatic Triple Negative Breast Cancer (TNBC). On vascularized tumors, the limited penetration of nanocarriers (<50-80 µm) combined with the short range of α-particles (40-100 µm) may, however, result in only partial tumor irradiation, compromising efficacy. Utilizing the α-particle emitter Actinium-225 (225Ac), we studied how the therapeutic potential of a general delivery strategy using nanometer-sized engineered liposomes was affected by two key transport-driven properties: (1) the release from liposomes, when in the tumor interstitium, of the highly diffusing 225Ac-DOTA that improves the uniformity of tumor irradiation by α-particles and (2) the adhesion of liposomes on the tumors' ECM that increases liposomes' time-integrated concentrations within tumors and, therefore, the tumor-delivered radioactivities. METHODS: On an orthotopic MDA-MB-231 TNBC murine model forming spontaneous metastases, we evaluated the maximum tolerated dose (MTD), biodistributions, and control of tumor growth and/or spreading after administration of 225Ac-DOTA-encapsulating liposomes, with different combinations of the two transport-driven properties. RESULTS: At 83% of MTD, 225Ac-DOTA-encapsulating liposomes with both properties (1) eliminated formation of spontaneous metastases and (2) best inhibited the progression of orthotopic xenografts, compared to liposomes lacking one or both properties. These findings were primarily affected by the extent of uniformity of the intratumoral microdistributions of 225Ac followed by the overall tumor uptake of radioactivity. At the MTD, long-term toxicities were not detected 9.5 months post administration. CONCLUSION: Our findings demonstrate the potential of a general, transport-driven strategy enabling more uniform and prolonged solid tumor irradiation by α-particles without cell-specific targeting.

Molecular and functional imaging insights into the role of hypoxia in cancer aggression.

Hypoxia in cancers has evoked significant interest since 1955 when Thomlinson and Gray postulated the presence of hypoxia in human lung cancers, based on the observation of necrosis occurring at the diffusion limit of oxygen from the nearest blood vessel, and identified the implication of these observations for radiation therapy. Coupled with discoveries in 1953 by Gray and others that anoxic cells were resistant to radiation damage, these observations have led to an entire field of research focused on exploiting oxygenation and hypoxia to improve the outcome of radiation therapy. Almost 65 years later, tumor heterogeneity of nearly every parameter measured including tumor oxygenation, and the dynamic landscape of cancers and their microenvironments are clearly evident, providing a strong rationale for cancer personalized medicine. Since hypoxia is a major cause of extracellular acidosis in tumors, here, we have focused on the applications of imaging to understand the effects of hypoxia in tumors and to target hypoxia in theranostic strategies. Molecular and functional imaging have critically important roles to play in personalized medicine through the detection of hypoxia, both spatially and temporally, and by providing new understanding of the role of hypoxia in cancer aggressiveness. With the discovery of the hypoxia-inducible factor (HIF), the intervening years have also seen significant progress in understanding the transcriptional regulation of hypoxia-induced genes. These advances have provided the ability to silence HIF and understand the associated molecular and functional consequences to expand our understanding of hypoxia and its role in cancer aggressiveness. Most recently, the development of hypoxia-based theranostic strategies that combine detection and therapy are further establishing imaging-based treatment strategies for precision medicine of cancer.

The Impact of the COVID-19 Pandemic on the Radiology Research Enterprise: Radiology Scientific Expert Panel.

The current coronavirus disease 2019 (COVID-19) crisis continues to grow and has resulted in marked changes to clinical operations. In parallel with clinical preparedness, universities have shut down most scientific research activities. Radiology researchers are currently grappling with these challenges that will continue to affect current and future imaging research. The purpose of this article is to describe the collective experiences of a diverse international group of academic radiology research programs in managing their response to the COVID-19 pandemic. The acute response at six distinct institutions will be described first, exploring common themes, challenges, priorities, and practices. This will be followed by reflections about the future of radiology research in the wake of the COVID-19 pandemic.

MRI and MRS of intact perfused cancer cell metabolism, invasion, and stromal cell interactions.

Because of the spatial and temporal heterogeneities of cancers, technologies to investigate cancer cells and the consequences of their interactions with abnormal physiological environments, such as hypoxia and acidic extracellular pH, with stromal cells, and with the extracellular matrix, under controlled conditions, are valuable to gain insights into the functioning of cancers. These insights can lead to an understanding of why cancers invade and metastasize, and identify effective treatment strategies. Here we have provided an overview of the applications of MRI/MRS/MRSI to investigate intact perfused cancer cells, their metabolism and invasion, and their interactions with stromal cells and the extracellular matrix.

Translating preclinical MRI methods to clinical oncology.

The complexity of modern in vivo magnetic resonance imaging (MRI) methods in oncology has dramatically changed in the last 10 years. The field has long since moved passed its (unparalleled) ability to form images with exquisite soft-tissue contrast and morphology, allowing for the enhanced identification of primary tumors and metastatic disease. Currently, it is not uncommon to acquire images related to blood flow, cellularity, and macromolecular content in the clinical setting. The acquisition of images related to metabolism, hypoxia, pH, and tissue stiffness are also becoming common. All of these techniques have had some component of their invention, development, refinement, validation, and initial applications in the preclinical setting using in vivo animal models of cancer. In this review, we discuss the genesis of quantitative MRI methods that have been successfully translated from preclinical research and developed into clinical applications. These include methods that interrogate perfusion, diffusion, pH, hypoxia, macromolecular content, and tissue mechanical properties for improving detection, staging, and response monitoring of cancer. For each of these techniques, we summarize the 1) underlying biological mechanism(s); 2) preclinical applications; 3) available repeatability and reproducibility data; 4) clinical applications; and 5) limitations of the technique. We conclude with a discussion of lessons learned from translating MRI methods from the preclinical to clinical setting, and a presentation of four fundamental problems in cancer imaging that, if solved, would result in a profound improvement in the lives of oncology patients. Level of Evidence: 5 Technical Efficacy: Stage 3 J. Magn. Reson. Imaging 2019;50:1377-1392.

Molecular causes of elevated phosphoethanolamine in breast and pancreatic cancer cells.

Elevated phosphoethanolamine (PE) is frequently observed in MRS studies of human cancers and xenografts. The role of PE in cell survival and the molecular causes underlying this increase are, however, relatively underexplored. In this study, we investigated the roles of ethanolamine kinases (Etnk-1 and 2) and choline kinases (Chk-α and ß) in contributing to increased PE in human breast and pancreatic cancer cells. We investigated the effect of silencing Etnk-1 and Etnk-2 on cell viability as a potential therapeutic strategy. Both breast and pancreatic cancer cells showed higher PE compared with their nonmalignant counterparts. We identified Etnk-1 as a major cause of the elevated PE levels in these cancer cells, with little or no contribution from Chk-α, Chk-ß, or Etnk-2. The increase of PE observed in pancreatic cancer cells in culture was replicated in the corresponding tumor xenografts. Downregulation of Etnk-1 with siRNA resulted in cell cytotoxicity that correlated with PE levels in breast and pancreatic cancer cells. Etnk-1 may provide a potential therapeutic target in breast and pancreatic cancers.

Lymphatic endothelial cells actively regulate prostate cancer cell invasion.

Lymphatic vessels serve as the primary route for metastatic spread to lymph nodes. However, it is not clear how interactions between cancer cells and lymphatic endothelial cells (LECs), especially within hypoxic microenvironments, affect the invasion of cancer cells. Here, using an MR compatible cell perfusion assay, we investigated the role of LEC-prostate cancer (PCa) cell interaction in the invasion and degradation of the extracellular matrix (ECM) by two human PCa cell lines, PC-3 and DU-145, under normoxia and hypoxia, and determined the metabolic changes that occurred under these conditions. We observed a significant increase in the invasion of ECM by invasive PC-3 cells, but not poorly invasive DU-145 cells when human dermal lymphatic microvascular endothelial cells (HMVEC-dlys) were present. Enhanced degradation of ECM by PC-3 cells in the presence of HMVEC-dlys identified interactions between HMVEC-dlys and PCa cells influencing cancer cell invasion. The enhanced ECM degradation was partly attributed to increased MMP-9 enzymatic activity in PC-3 cells when HMVEC-dlys were in close proximity. Significantly higher uPAR and MMP-9 expression levels observed in PC-3 cells compared to DU-145 cells may be one mechanism for increased invasion and degradation of matrigel by these cells irrespective of the presence of HMVEC-dlys. Hypoxia significantly decreased invasion by PC-3 cells, but this decrease was significantly attenuated when HMVEC-dlys were present. Significantly higher phosphocholine was observed in invasive PC-3 cells, while higher glycerophosphocholine was observed in DU-145 cells. These metabolites were not altered in the presence of HMVEC-dlys. Significantly increased lipid levels and lipid droplets were observed in PC-3 and DU-145 cells under hypoxia reflecting an adaptive survival response to oxidative stress. These results suggest that in vivo, invasive cells in or near lymphatic endothelial cells are likely to be more invasive and degrade the ECM to influence the metastatic cascade. Copyright © 2016 John Wiley & Sons, Ltd.

Targeting choline phospholipid metabolism: GDPD5 and GDPD6 silencing decrease breast cancer cell proliferation, migration, and invasion.

Abnormal choline phospholipid metabolism is associated with oncogenesis and tumor progression. We have investigated the effects of targeting choline phospholipid metabolism by silencing two glycerophosphodiesterase genes, GDPD5 and GDPD6, using small interfering RNA (siRNA) in two breast cancer cell lines, MCF-7 and MDA-MB-231. Treatment with GDPD5 and GDPD6 siRNA resulted in significant increases in glycerophosphocholine (GPC) levels, and no change in the levels of phosphocholine or free choline, which further supports their role as GPC-specific regulators in breast cancer. The GPC levels were increased more than twofold during GDPD6 silencing, and marginally increased during GDPD5 silencing. DNA laddering was negative in both cell lines treated with GDPD5 and GDPD6 siRNA, indicating absence of apoptosis. Treatment with GDPD5 siRNA caused a decrease in cell viability in MCF-7 cells, while GDPD6 siRNA treatment had no effect on cell viability in either cell line. Decreased cell migration and invasion were observed in MDA-MB-231 cells treated with GDPD5 or GDPD6 siRNA, where a more pronounced reduction in cell migration and invasion was observed under GDPD5 siRNA treatment as compared with GDPD6 siRNA treatment. In conclusion, GDPD6 silencing increased the GPC levels in breast cancer cells more profoundly than GDPD5 silencing, while the effects of GDPD5 silencing on cell viability/proliferation, migration, and invasion were more severe than those of GDPD6 silencing. Our results suggest that silencing GDPD5 and GDPD6 alone or in combination may have potential as a new molecular targeting strategy for breast cancer treatment. Copyright © 2016 John Wiley & Sons, Ltd.

Effect of alginate microencapsulation on the catalytic efficiency and in vitro enzyme-prodrug therapeutic efficacy of cytosine deaminase and of recombinant E. coli expressing cytosine deaminase.

Cytosine deaminase (CD) catalyses the enzymatic conversion of the non-toxic prodrug 5-fluorocytosine (5-FC) to the potent chemotherapeutic form, 5-fluorouracil (5-FU). Intratumoral delivery of CD localises chemotherapy dose while reducing systemic toxicity. Encapsulation in biocompatible microcapsules immunoisolates CD and protects it from degradation. We report on the effect of alginate encapsulation on the catalytic and functional activity of isolated CD and recombinant E. coli engineered to express CD (E. coli(CD)). Alginate microcapsules containing either CD or Escherichia coli(CD) were prepared using ionotropic gelation. Conversion of 5-FC to 5-FU was quantitated in unencapsulated and encapsulated CD/E. coli(CD) using spectrophotometry, with a slower rate of conversion observed following encapsulation. Both encapsulated CD/5-FC and E. coli(CD)/5-FC resulted in cell kill and reduced proliferation of 9 L rat glioma cells, which was comparable to direct 5-FU treatment. Our results show that encapsulation preserves the therapeutic potential of CD and E. coli(CD) is equally effective for enzyme-prodrug therapy.

MALDI-Mass Spectrometric Imaging Revealing Hypoxia-Driven Lipids and Proteins in a Breast Tumor Model.

Hypoxic areas are a common feature of rapidly growing malignant tumors and their metastases and are typically spatially heterogeneous. Hypoxia has a strong impact on tumor cell biology and contributes to tumor progression in multiple ways. To date, only a few molecular key players in tumor hypoxia, such as hypoxia-inducible factor-1 (HIF-1), have been discovered. The distribution of biomolecules is frequently heterogeneous in the tumor volume and may be driven by hypoxia and HIF-1α. Understanding the spatially heterogeneous hypoxic response of tumors is critical. Mass spectrometric imaging (MSI) provides a unique way of imaging biomolecular distributions in tissue sections with high spectral and spatial resolution. In this paper, breast tumor xenografts grown from MDA-MB-231-HRE-tdTomato cells, with a red fluorescent tdTomato protein construct under the control of a hypoxia response element (HRE)-containing promoter driven by HIF-1α, were used to detect the spatial distribution of hypoxic regions. We elucidated the 3D spatial relationship between hypoxic regions and the localization of lipids and proteins by using principal component analysis-linear discriminant analysis (PCA-LDA) on 3D rendered MSI volume data from MDA-MB-231-HRE-tdTomato breast tumor xenografts. In this study, we identified hypoxia-regulated proteins active in several distinct pathways such as glucose metabolism, regulation of actin cytoskeleton, protein folding, translation/ribosome, splicesome, the PI3K-Akt signaling pathway, hemoglobin chaperone, protein processing in endoplasmic reticulum, detoxification of reactive oxygen species, aurora B signaling/apoptotic execution phase, the RAS signaling pathway, the FAS signaling pathway/caspase cascade in apoptosis, and telomere stress induced senescence. In parallel, we also identified colocalization of hypoxic regions and various lipid species such as PC(16:0/18:0), PC(16:0/18:1), PC(16:0/18:2), PC(16:1/18:4), PC(18:0/18:1), and PC(18:1/18:1), among others. Our findings shed light on the biomolecular composition of hypoxic tumor regions, which may be responsible for a given tumor's resistance to radiation or chemotherapy.

Choline kinase-α protein and phosphatidylcholine but not phosphocholine are required for breast cancer cell survival.

High levels of total choline and phosphocholine (PC) are consistently observed in aggressive cancers. Choline kinase (Chk) catalyzes choline phosphorylation to produce PC in phosphatidylcholine (PtdCho) biosynthesis. PtdCho is the most abundant phospholipid in eukaryotic cell membranes and plays a dual role as the structural component of membranes and as a substrate to produce lipid second messengers such as phosphatidic acid and diacylglycerol. Chk-α, but not Chk-ß, is overexpressed in various cancers, and is closely associated with tumor progression and invasiveness. We have previously shown that downregulation of mRNA using small interfering RNA (siRNA) against Chk-α (siRNA-Chk) or Chk short hairpin RNA, and the resultant decrease of Chk-α protein levels, significantly reduced proliferation in breast cancer cells and tumors. A novel potent and selective small-molecule Chk-α inhibitor, V-11-0711, that inhibits the catalytic activity of Chk has recently been developed. Here, we used triple negative MDA-MB-231 and SUM149 breast cancer cells to further investigate the role of Chk-α in cancer, by examining Chk-α protein levels, cell viability/proliferation, choline phospholipid and lipid metabolism, lipid droplet formation, and apoptosis, following treatment with V-11-0711. Under the conditions used in this study, treatment with V-11-0711 significantly decreased PC levels but did not reduce cell viability as long as Chk-α protein and PtdCho levels were not reduced, suggesting that Chk-α protein and PtdCho, but not PC, may be crucial for breast cancer cell survival. These data also support the approach of antitumor strategies that destabilize Chk-α protein or downregulate PtdCho in breast cancer treatment.

HIF-dependent antitumorigenic effect of antioxidants in vivo.

The antitumorigenic activity of antioxidants has been presumed to arise from their ability to squelch DNA damage and genomic instability mediated by reactive oxygen species (ROS). Here, we report that antioxidants inhibited three tumorigenic models in vivo. Inhibition of a MYC-dependent human B lymphoma model was unassociated with genomic instability but was linked to diminished hypoxia-inducible factor (HIF)-1 levels in a prolyl hydroxylase 2 and von Hippel-Lindau protein-dependent manner. Ectopic expression of an oxygen-independent, stabilized HIF-1 mutant rescued lymphoma xenografts from inhibition by two antioxidants: N-acetylcysteine and vitamin C. These findings challenge the paradigm that antioxidants diminish tumorigenesis primarily through decreasing DNA damage and mutations and provide significant support for a key antitumorigenic effect of diminishing HIF levels.

The HOXB7 protein renders breast cancer cells resistant to tamoxifen through activation of the EGFR pathway.

Multiple factors including long-term treatment with tamoxifen are involved in the development of selective estrogen receptor (ER) modulator resistance in ERα-positive breast cancer. Many underlying molecular events that confer resistance are known but a unifying theme is yet to be revealed. In this report, we provide evidence that HOXB7 overexpression renders MCF-7 cells resistant to tamoxifen via cross-talk between receptor tyrosine kinases and ERα signaling. HOXB7 is an ERα-responsive gene. Extended treatment of MCF-7 cells with tamoxifen resulted in progressively increasing levels of HOXB7 expression, along with EGFR and EGFR ligands. Up-regulation of EGFR occurs through direct binding of HOXB7 to the EGFR promoter, enhancing transcriptional activity. Finally, higher expression levels of HOXB7 in the tumor significantly correlated with poorer disease-free survival in ERα-positive patients with breast cancer on adjuvant tamoxifen monotherapy. These studies suggest that HOXB7 acts as a key regulator, orchestrating a major group of target molecules in the oncogenic hierarchy. Functional antagonism of HOXB7 could circumvent tamoxifen resistance.