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Local hypoxia occurs in most solid tumors and is associated with aggressive disease and therapy resistance. Widespread changes in gene expression play a critical role in the biological response to hypoxia. However, most research has focused on hypoxia‐inducible genes as opposed to those that are decreased in hypoxia. We demonstrate that chromatin accessibility is decreased in hypoxia, predominantly at gene promoters and specific pathways are impacted including DNA repair, splicing, and the R‐loop interactome. One of the genes with decreased chromatin accessibility in hypoxia was DDX5, encoding the RNA helicase, DDX5, which showed reduced expression in various cancer cell lines in hypoxic conditions, tumor xenografts, and in patient samples with hypoxic tumors. Most interestingly, we found that when DDX5 is rescued in hypoxia, replication stress and R‐loop levels accumulate further, demonstrating that hypoxia‐mediated repression of DDX5 restricts R‐loop accumulation. Together these data support the hypothesis that a critical part of the biological response to hypoxia is the repression of multiple R‐loop processing factors; however, as shown for DDX5, their role is specific and distinct.

Abstract Background Clear cell renal cell carcinoma (ccRCC), the predominant subtype of kidney cancer, possesses characteristic alterations to multiple metabolic pathways, including the accumulation of cytosolic lipid droplets. However, the pathways that drive lipid droplet accumulation in ccRCC cells and their importance to cancer biology remain poorly understood. Methods We sought to identify the carbon sources necessary for lipid droplet accumulation using Oil red O staining and isotope-tracing lipidomics. The role of the acyl-CoA synthetase (ACSL) family members, an important group of lipid metabolic enzymes, was investigated using siRNA and drug mediated inhibition. CTB and XTT assays were performed to determine the effect of ACSL3 knockdown and lipid starvation on ccRCC cell viability and shRNA was used to study the effect of ACSL3 in an orthotopic mouse model. The relationship between ferroptosis susceptibility of ccRCC and ACSL3 controlled lipid metabolism was examined using CTB and FACS-based assays. The importance of 5-LOX in ferroptosis susceptibility in ccRCC was shown with XTT survival assays, and the expression level and predictive value of 5-LOX in TCGA ccRCC data was assessed. Results We found that ccRCC cells obtain the necessary substrates for lipid droplet accumulation by metabolizing exogenous serum derived lipids and not through de novo lipogenesis. We show that this metabolism of exogenous fatty acids into lipid droplets requires the enzyme acyl-CoA synthetase 3 (ACSL3) and not other ACSL family proteins. Importantly, genetic or pharmacologic suppression of ACSL3 is cytotoxic to ccRCC cells in vitro and causes a reduction of tumor weight in an orthotopic mouse model. Conversely, ACSL3 inhibition decreases the susceptibility of ccRCC cells to ferroptosis, a non-apoptotic form of cell death involving lipid peroxidation. The sensitivity of ccRCC to ferroptosis is also highly dependent on the composition of exogenous fatty acids and on 5-lipoxygenase (5-LOX), a leukotriene producing enzyme which produces lipid peroxides that have been implicated in other cancers but not in ccRCC. Conclusions ACSL3 regulates the accumulation of lipid droplets in ccRCC and is essential for tumor growth. In addition, ACSL3 also modulates ferroptosis sensitivity in a manner dependent on the composition of exogenous fatty acids. Both functions of ACSL3 could be exploited for ccRCC therapy.

Hypoxia plays a critical role in tumor progression including invasion and metastasis. Here, the authors screened several hypoxia inducible genes and identified the oncogenic role of MAFF in breast cancer metastasis and that it activates IL11/STAT3 pathway.

Abstract Background Tumor hypoxia is a characteristic of paramount importance due to low oxygenation levels in tissue negatively correlating with resistance to traditional therapies. The ability to noninvasively identify such could provide for personalized treatment(s) and enhance survival rates. Accordingly, we recently developed an NIR fluorescent hypoxia‐sensitive smart probe (NO2‐Rosol) for identifying hypoxia via selectively imaging nitroreductase (NTR) activity, which could correlate to oxygen deprivation levels in cells, thereby serving as a proxy. We demonstrated proof of concept by subjecting a glioblastoma (GBM) cell line to extreme stress by evaluating such under radiobiological hypoxic (pO2 ≤ ~0.5%) conditions, which is a far cry from representative levels for hypoxia for brain glioma (pO2 = ~1.7%) which fluctuate little from physiological hypoxic (pO2 = 1.0‐3.0%) conditions. Aim We aimed to evaluate the robustness, suitability, and feasibility of NO2‐Rosol for imaging hypoxia in vitro and in vivo via assessing NTR activity in diverse GBM models under relevant oxygenation levels (pO2 = 2.0%) within physiological hypoxic conditions that mimic oxygenation levels in GBM tumor tissue in the brain. Methods We evaluated multiple GBM cell lines to determine their relative sensitivity to oxygenation levels via measuring carbonic anhydrase IX (CAIX) levels, which is a surrogate marker for indirectly identifying hypoxia by reporting on oxygen deprivation levels and upregulated NTR activity. We evaluated for hypoxia via measuring NTR activity when employing NO2‐Rosol in in vitro and tumor hypoxia imaging studies in vivo. Results The GBM39 cell line demonstrated the highest CAIX expression under hypoxic conditions representing that of GBM in the brain. NO2‐Rosol displayed an 8‐fold fluorescence enhancement when evaluated in GBM39 cells (pO2 = 2.0%), thereby establishing its robustness and suitability for imaging hypoxia under relevant physiological conditions. We demonstrated the feasibility of NO2‐Rosol to afford tumor hypoxia imaging in vivo via it demonstrating a tumor‐to‐background of 5 upon (i) diffusion throughout, (ii) bioreductive activation by NTR activity in, and (iii) retention within, GBM39 tumor tissue. Conclusion We established the robustness, suitability, and feasibility of NO2‐Rosol for imaging hypoxia under relevant oxygenation levels in vitro and in vivo via assessing NTR activity in GBM39 models.

Summary: The importance of innate immunity in cancer is increasingly being recognized with recent reports suggesting tumor cell-intrinsic intracellular functions for innate immunity proteins. However, such functions are often poorly understood, and it is unclear whether these are affected by patient-specific mutations. Here, we show that C4b-binding protein alpha chain (C4BPA), typically thought to reside in the extracellular space, is expressed intracellularly in cancer cells, where it interacts with the NF-κB family member RelA and regulates apoptosis. Interestingly, intracellular C4BPA expression is regulated in a stress- and mutation-dependent manner and C4BPA mutations are associated with improved cancer survival outcome. Using cell lines harboring patient-specific C4BPA mutations, we show that increasing intracellular C4BPA levels correlate with sensitivity to oxaliplatin-induced apoptosis in vitro and in vivo. Mechanistically, sensitive C4BPA mutants display increased IκBα expression and increased inhibitory IκBα-RelA complex stability. These data suggest a non-canonical intracellular role for C4BPA in regulating NF-κB-dependent apoptosis.

Irradiation and temozolomide (TMZ) chemotherapy are the current standard treatments for glioblastoma multiforme (GBM), but they are associated with toxicity and limited efficacy. Recently, these standard therapies have been used to enhance immunotherapy against GBM. Immunotherapy using the anti-CD47 (immune checkpoint inhibitor) treatment has shown promise in treating multiple tumor types, including GBM. The goal of this current work was to test whether irradiation or TMZ chemotherapy could enhance anti-CD47 treatment against GBM. Our results showed that irradiation and TMZ each significantly enhanced anti-CD47-mediated phagocytosis of GBM cells in vitro . Furthermore, mice engrafted with human GBM that received anti-CD47 combined with focal irradiation or TMZ treatment showed a significant increase in the survival rate compared to those that received a single treatment. The tumor growth in mice that received both anti-CD47 and irradiation was significantly less than that of groups that received either anti-CD47 or focal irradiation. The results from this study may support future use of anti-CD47 treatment in combination with irradiation or chemotherapy to enhance the therapeutic efficacy of GBM treatment.

Cellular heterogeneity in cancer is complex and difficult to study. Here, the authors introduce Protein-indexed Assay of Transposase Accessible Chromatin (Pi-ATAC), which combines single cell chromatin and proteomic profiling to provide deep insight into the tumor microenvironment, and reveal the role of hypoxia in shaping the regulome of a subset of breast cancer cells in vivo.

Radiotherapy can enhance the antitumour immune response. Here, the authors show that resistance to radiation in breast cancer cells can be due to Axl expression that suppresses antigen presentation though MHCI, promotes NF-κB signalling, and enhances cytokine release promoting a suppressive myeloid microenvironment.

Summary: Complement-mediated cytotoxicity may act as a selective pressure for tumor overexpression of complement regulators. We hypothesize that the same selective pressure could lead to complement alterations at the genetic level. We find that, when analyzed as a pathway, mutations in complement genes occur at a relatively high frequency and are associated with changes in overall survival across a number of cancer types. Analysis of pathways expressed in patients with complement mutations that are associated with poor overall survival reveals crosstalk between complement and hypoxia in colorectal cancer. The importance of this crosstalk is highlighted by two key findings: hypoxic signaling is increased in tumors harboring complement mutations, and hypoxic tumor cells are resistant to complement-mediated cytotoxicity due, in part, to hypoxia-induced expression of complement regulator CD55. The range of strategies employed by tumors to dysregulate the complement system testifies to the importance of this pathway in tumor progression. : Mutations in the complement system are prevalent across cancers. Olcina et al. find that colorectal cancers with complement component mutations are associated with increased hypoxic signaling and poor overall survival outcomes. Hypoxia-induced expression of complement regulator CD55 controls complement-mediated cytotoxicity. Keywords: innate immunity, cancer, mutations, hypoxia, complement system, complement-mediated cytotoxicity

There has been strong demand for the development of an accurate but simple method to assess the freshness of food. In this study, we demonstrated a system to determine food freshness by analyzing the spectral response from a portable visible/near-infrared (VIS/NIR) spectrometer using the Convolutional Neural Network (CNN)-based machine learning algorithm. Spectral response data from salmon, tuna, and beef incubated at 25 °C were obtained every minute for 30 h and then categorized into three states of “fresh”, “likely spoiled”, and “spoiled” based on time and pH. Using the obtained spectral data, a CNN-based machine learning algorithm was built to evaluate the freshness of experimental objects. In addition, a CNN-based machine learning algorithm with a shift-invariant feature can minimize the effect of the variation caused using multiple devices in a real environment. The accuracy of the obtained machine learning model based on the spectral data in predicting the freshness was approximately 85% for salmon, 88% for tuna, and 92% for beef. Therefore, our study demonstrates the practicality of a portable spectrometer in food freshness assessment.

Long believed to be a byproduct of malignant transformation, reprogramming of cellular metabolism is now recognized as a driving force in tumorigenesis. In clear cell renal cell carcinoma (ccRCC), frequent activation of HIF signaling induces a metabolic switch that promotes tumorigenesis. Here, we demonstrate that PGC-1α, a central regulator of energy metabolism, is suppressed in VHL-deficient ccRCC by a HIF/Dec1-dependent mechanism. In VHL wild-type cells, PGC-1α suppression leads to decreased expression of the mitochondrial transcription factor Tfam and impaired mitochondrial respiration. Conversely, PGC-1α expression in VHL-deficient cells restores mitochondrial function and induces oxidative stress. ccRCC cells expressing PGC-1α exhibit impaired tumor growth and enhanced sensitivity to cytotoxic therapies. In patients, low levels of PGC-1α expression are associated with poor outcome. These studies demonstrate that suppression of PGC-1α recapitulates key metabolic phenotypes of ccRCC and highlight the potential of targeting PGC-1α expression as a therapeutic modality for the treatment of ccRCC.

The p53 tumor suppressor plays a key role in maintaining cellular integrity. In response to diverse stress signals, p53 can trigger apoptosis to eliminate damaged cells or cell-cycle arrest to enable cells to cope with stress and survive. However, the transcriptional networks underlying p53 pro-survival function are incompletely understood. Here, we show that in oncogenic-Ras-expressing cells, p53 promotes oxidative phosphorylation (OXPHOS) and cell survival upon glucose starvation. Analysis of p53 transcriptional activation domain mutants reveals that these responses depend on p53 transactivation function. Using gene expression profiling and ChIP-seq analysis, we identify several p53-inducible fatty acid metabolism-related genes. One such gene, Acad11, encoding a protein involved in fatty acid oxidation, is required for efficient OXPHOS and cell survival upon glucose starvation. This study provides new mechanistic insight into the pro-survival function of p53 and suggests that targeting this pathway may provide a strategy for therapeutic intervention based on metabolic perturbation.

Radiotherapy (RT) is a localized therapy that is highly effective in killing primary tumor cells located within the field of the radiation beam. We present evidence that irradiation of breast tumors can attract migrating breast cancer cells. Granulocyte-macrophage colony stimulating factor (GM-CSF) produced by tumor cells in response to radiation stimulates the recruitment of migrating tumor cells to irradiated tumors, suggesting a mechanism of tumor recurrence after radiation facilitated by transit of unirradiated, viable circulating tumor cells to irradiated tumors. Data supporting this hypothesis are presented through in vitro invasion assays and in vivo orthotopic models of breast cancer. Our work provides a mechanism for tumor recurrence in which RT attracts cells outside the radiation field to migrate to the site of treatment.

Previous studies have suggested that tumor hypoxia could be exploited for cancer gene therapy. Using hypoxia-responsive elements derived from the human vascular endothelial growth factor gene, we have generated vectors expressing a bacterial nitroreductase. (20NTR) gene that can activate the anticancer prodrug CB1954. Stable transfectants of human HT1080 tumor cells with hypoxia-inducible vectors were established with G418 selection. Hypoxic induction of NTR protein correlated with increased sensitivity to in vitro exposure of HT 1080 cells to the prodrug. Growth delay assays were performed with established tumor xenografts derived from the same cells to detect the in vivo efficacy of CB1954 conversion to its cytotoxic form. Significant antitumor effects were achieved with intraperitoneal injections of CB1954 both in tumors that express NTR constitutively or with a hypoxia-inducible promoter. In addition, respiration of 10% O2 increased tumor hypoxia in vivo and enhanced the antitumor effects. Taken together, these results demonstrate that hypoxia-inducible vectors may be useful for tumor-selective gene therapy, although the problem of delivery of the vector to the tumors, particularly to the hypoxic cells in the tumors, is not addressed by these studies.

INTRODUCTION:Hypoxia-inducible factor (HIF)-1α, part of the heterodimeric transcription factor that mediates the cellular response to hypoxia, is critical for the expression of multiple angiogenic growth factors, cell motility, and the recruitment of endothelial progenitor cells. Inhibition of the oxygen-dependent negative regulator of HIF-1α, prolyl hydroxylase domain-2 (PHD-2), leads to increased HIF-1α and mimics various cellular and physiological responses to hypoxia. The roles of PHD-2 in the epidermis and dermis have not been clearly defined in wound healing. METHODS:Epidermal and dermal specific PHD-2 knockout (KO) mice were developed in a C57BL/6J (wild type) background by crossing homozygous floxed PHD-2 mice with heterozygous K14-Cre mice and heterozygous Col1A2-Cre-ER mice to get homozygous floxed PHD-2/heterozygous K14-Cre and homozygous floxed PHD-2/heterozygous floxed Col1A2-Cre-ER mice, respectively. Ten to twelve-week-old PHD-2 KO and wild type (WT) mice were subjected to wounding and ischemic pedicle flap model. The amount of healing was grossly quantified with ImageJ software. Western blot and qRT-PCR was run on protein and RNA from primary cells cultured in vitro. RESULTS:qRT-PCR demonstrated a significant decrease of PHD-2 in keratinocytes and fibroblasts derived from tissue specific KO mice relative to control mice (*p

The intervertebral disc (IVD) is one of the largest avascular organs in vertebrates. The nucleus pulposus (NP), a highly hydrated and proteoglycan-enriched tissue, forms the inner portion of the IVD. The NP is surrounded by a multi-lamellar fibrocartilaginous structure, the annulus fibrosus (AF). This structure is covered superior and inferior side by cartilaginous endplates (CEP). The NP is a unique tissue within the IVD as it results from the differentiation of notochordal cells, whereas, AF and CEP derive from the sclerotome. The hypoxia inducible factor-1α (HIF-1α) is expressed in NP cells but its function in NP development and homeostasis is largely unknown. We thus conditionally deleted HIF-1α in notochordal cells and investigated how loss of this transcription factor impacts NP formation and homeostasis at E15.5, birth, 1 and 4 months of age, respectively. Histological analysis, cell lineage studies, and TUNEL assay were performed. Morphologic changes of the mutant NP cells were identified as early as E15.5, followed, postnatally, by the progressive disappearance and replacement of the NP with a novel tissue that resembles fibrocartilage. Notably, lineage studies and TUNEL assay unequivocally proved that NP cells did not transdifferentiate into chondrocyte-like cells but they rather underwent massive cell death, and were completely replaced by a cell population belonging to a lineage distinct from the notochordal one. Finally, to evaluate the functional consequences of HIF-1α deletion in the NP, biomechanical testing of mutant IVD was performed. Loss of the NP in mutant mice significantly reduced the IVD biomechanical properties by decreasing its ability to absorb mechanical stress. These findings are similar to the changes usually observed during human IVD degeneration. Our study thus demonstrates that HIF-1α is essential for NP development and homeostasis, and it raises the intriguing possibility that this transcription factor could be involved in IVD degeneration in humans.

Original citation: J Clin Invest. 2023;133(23):e168277. https://doi.org/10.n72/JCI168277. During the preparation of this manuscript, the dose for PMX205 used for in vitro experiments was stated incorrectly in the legend for Figure [...]

Bone marrow is a highly heterogeneous and vascularized tissue. The various cell types populating the bone marrow extensively communicate with each other, and cell to cell cross talk is likely to be essential for proper bone development and homeostasis. In particular, the existence of osteogenesis and angiogenesis coupling has been recently proposed. Despite its high degree of vascularization, a gradient of oxygenation is present in the bone marrow, and the endosteal surface of cortical bone appears to be among the most hypoxic areas in the body. Oxygen (O2) is both an essential metabolic substrate and a regulatory signal that is in charge of a specific genetic program. An important component of this program is the family of transcription factors known as hypoxia-inducible factors or HIFs. In this Perspective, we will summarize our current knowledge about the role of the HIF signaling pathway in controlling bone development and homeostasis, and especially in regulating the crosstalk between osteoblasts, progenitor cells, and bone marrow blood vessels.

Tumor hypoxia plays a crucial role in tumorigenesis. Under hypoxia, hypoxia-inducible factor 1α (HIF-1α) regulates activation of genes promoting malignant progression. Under normoxia, HIF-1α is hydroxylated on prolines 402 and 564 and is targeted for ubiquitin-mediated degradation by interacting with the von Hippel-Lindau protein complex (pVHL). We have developed a novel method of studying the interaction between HIF-1α and pVHL using the split firefly luciferase complementation-based bioluminescence system in which HIF-1α and pVHL are fused to amino-terminal and carboxy-terminal fragments of the luciferase, respectively. We demonstrate that hydroxylation-dependent interaction between the HIF-1α and pVHL leads to complementation of the two luciferase fragments, resulting in bioluminescence in vitro and in vivo. Complementation-based bioluminescence is diminished when mutant pVHLs with decreased affinity for binding HIF-1α are used. This method represents a new approach for studying interaction of proteins involved in the regulation of protein degradation.

Reporter gene techniques have been applied toward studying the physiologic phenomena associated with tumor hypoxia, a negative prognostic indicator. The purpose of this study was to assess the potential adverse effects of hypoxic conditions on the effectiveness of four commonly used reporter genes: Renilla luciferase, monomeric red fluorescent protein, thymidine kinase, and lacZ . Tumor-forming A375 cells expressing a trifusion reporter consisting of Renilla luciferase, monomeric red fluorescent protein, and thymidine kinase were subjected to decreasing oxygen tensions and assayed for reporter expression and activity. A375 cells expressing β-galactosidase were similarly exposed to hypoxia, with activity of the reporter monitored by cleavage of the fluorescent substrate 7-hydroxy-9 H -(1, 3-dichloro-9, 9-dimethylacridin-2-one)-β-galactoside (DDAOG). Generation of signal in in vivo tumor models expressing bioluminescent or β-galactosidase reporters were also examined over the course of hypoxic stresses, either by tumor clamping or the antivascular agent 5, 6-dimethylxanthenone-4-acetic acid (DMXAA). Our findings indicate that bioluminescent and fluorescent reporter activity are decreased under hypoxia despite minimal variations in protein production, whereas β-galactosidase reporter activity per unit protein was unchanged. These results demonstrate that combining β-galactosidase with the DDAOG optical probe may be a robust reporter system for the in vivo study of tumor hypoxia.

An immunosuppressive microenvironment causes poor tumor T cell infiltration and is associated with reduced patient overall survival in colorectal cancer. How to improve treatment responses in these tumors is still a challenge. Using an integrated screening approach to identify cancer-specific vulnerabilities, we identified complement receptor C5aR1 as a druggable target, which when inhibited improved radiotherapy, even in tumors displaying immunosuppressive features and poor [CD8.sup.+] T cell infiltration. While C5aR1 is well-known for its role in the immune compartment, we found that C5aR1 is also robustly expressed on malignant epithelial cells, highlighting potential tumor cell-specific functions. C5aR1 targeting resulted in increased NF-[kappa]B-dependent apoptosis specifically in tumors and not normal tissues, indicating that, in malignant cells, C5aR1 primarily regulated cell fate. Collectively, these data revealed that increased complement gene expression is part of the stress response mounted by irradiated tumors and that targeting C5aR1 could improve radiotherapy, even in tumors displaying immunosuppressive features., Introduction The composition of the tumor microenvironment (TME) affects treatment responses in cancer (1-3). Immunosuppressive TME features, which act as a barrier to extensive [CD8.sup.+] T cell infiltration typically characterize [...]

The complement system is an innate immune pathway typically thought of as part of the first line of defence against "non-self" species. In the context of cancer, complement has been described to have an active role in facilitating cancer-associated processes such as increased proliferation, angiogenesis and migration. Several cellular members of the tumour microenvironment express and/or produce complement proteins locally, including tumour cells. Dysregulation of the complement system has been reported in numerous tumours and increased expression of complement activation fragments in cancer patient specimens correlates with poor patient prognosis. Importantly, genetic or pharmacological targeting of complement has been shown to reduce tumour growth in several cancer preclinical models, suggesting that complement could be an attractive therapeutic target. Hypoxia (low oxygen) is frequently found in solid tumours and has a profound biological impact on cellular and non-cellular components of the tumour microenvironment. In this review, we focus on hypoxia since this is a prevailing feature of the tumour microenvironment that, like increased complement, is typically associated with poor prognosis. Furthermore, interesting links between hypoxia and complement have been recently proposed but never collectively reviewed. Here, we explore how hypoxia alters regulation of complement proteins in different cellular components of the tumour microenvironment, as well as the downstream biological consequences of this regulation.

Here we introduce Protein-indexed Assay of Transposase Accessible Chromatin with sequencing (Pi-ATAC) that combines single-cell chromatin and proteomic profiling. In conjunction with DNA transposition, the levels of multiple cell surface or intracellular protein epitopes are recorded by index flow cytometry and positions in arrayed microwells, and then subject to molecular barcoding for subsequent pooled analysis. Pi-ATAC simultaneously identifies the epigenomic and proteomic heterogeneity in individual cells. Pi-ATAC reveals a casual link between transcription factor abundance and DNA motif access, and deconvolute cell types and states in the tumor microenvironment in vivo. We identify a dominant role for hypoxia, marked by HIF1α protein, in the tumor microvenvironment for shaping the regulome in a subset of epithelial tumor cells.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most metastatic and deadly cancers. Despite the clinical significance of metastatic spread, our understanding of molecular mechanisms that drive PDAC metastatic ability remains limited. By generating a genetically engineered mouse model of human PDAC, we uncover a transient subpopulation of cancer cells with exceptionally high metastatic ability. Global gene expression profiling and functional analyses uncovered the transcription factor BLIMP1 as a driver of PDAC metastasis. The highly metastatic PDAC subpopulation is enriched for hypoxia-induced genes, and hypoxia-mediated induction of BLIMP1 contributes to the regulation of a subset of hypoxia-associated gene expression programs. These findings support a model in which upregulation of BLIMP1 links microenvironmental cues to a metastatic stem cell character.Significance: PDAC is an almost uniformly lethal cancer, largely due to its tendency for metastasis. We define a highly metastatic subpopulation of cancer cells, uncover a key transcriptional regulator of metastatic ability, and define hypoxia as an important factor within the tumor microenvironment that increases metastatic proclivity. Cancer Discov; 7(10); 1184-99. ©2017 AACR.See related commentary by Vakoc and Tuveson, p. 1067This article is highlighted in the In This Issue feature, p. 1047.

It is well established that organs of future metastasis are not passive receivers of circulating tumour cells, but are instead selectively and actively modified by the primary tumour before metastatic spread has even occurred. Sowing the 'seeds' of metastasis requires the action of tumour-secreted factors and tumour-shed extracellular vesicles that enable the 'soil' at distant metastatic sites to encourage the outgrowth of incoming cancer cells. In this Review, we summarize the main processes and new mechanisms involved in the formation of the pre-metastatic niche.

The p53 tumor suppressor protein plays a critical role in orchestrating the genomic response to various stress signals by acting as a master transcriptional regulator. Differential gene activity is controlled by transcription factors but also dependent on the underlying chromatin structure, especially on covalent histone modifications. After screening different histone lysine methyltransferases and demethylases, we identified JMJD2B/KDM4B as a p53-inducible gene in response to DNA damage. p53 directly regulates JMJD2B gene expression by binding to a canonical p53-consensus motif in the JMJD2B promoter. JMJD2B induction attenuates the transcription of key p53 transcriptional targets including p21, PIG3 and PUMA, and this modulation is dependent on the catalytic capacity of JMJD2B. Conversely, JMJD2B silencing led to an enhancement of the DNA-damage driven induction of p21 and PIG3. These findings indicate that JMJD2B acts in an auto-regulatory loop by which p53, through JMJD2B activation, is able to influence its own transcriptional program. Functionally, exogenous expression of JMJD2B enhanced subcutaneous tumor growth of colon cancer cells in a p53-dependent manner, and genetic inhibition of JMJD2B impaired tumor growth in vivo. These studies provide new insights into the regulatory effect exerted by JMJD2B on tumor growth through the modulation of p53 target genes.

PurposePreclinical studies of hypoxia are generally done using ectopic xenograft tumors, which behave differently from human tumors. Our previous findings have shown that subcutaneously implanted lung tumors exhibit more hypoxia than their orthotopic implanted or spontaneous K-ras-induced counterparts. We hypothesize that differences in hypoxia are due to site-specific differences in vascularity and perfusion.ProceduresTo compare the presence and functionality of vessels in these tumor models, we studied vascular perfusion in vivo in real time.ResultsOrthotopically implanted and spontaneous K-ras-induced lung tumors showed elevated perfusion, demonstrating vasculature functionality. Little contrast agent uptake was observed within the subcutaneously implanted tumors, indicating vascular dysfunction. These findings were corroborated at the microscopic level with Hoechst 33342 and Meca-32 staining.ConclusionsFrom these observations, we concluded that differences in hypoxia in experimental models is related to vessel perfusion. Thus, appropriate selection of preclinical lung tumor models is essential for the study of hypoxia, angiogenesis and therapies targeting these phenomena.

Increasing evidence suggests that ionizing radiation therapy (RT) in combination with checkpoint immunotherapy is highly effective in treating a subset of cancers. To better understand the limited responses to this combination we analysed the genetic, microenvironmental, and immune factors in tumours derived from a transgenic breast cancer model. We identified two tumours with similar growth characteristics but different RT responses primarily due to an antitumour immune response. The combination of RT and checkpoint immunotherapy resulted in cures in the responsive but not the unresponsive tumours. Profiling the tumours revealed that the Axl receptor tyrosine kinase is overexpressed in the unresponsive tumours, and Axl knockout resulted in slower growth and increased radiosensitivity. These changes were associated with a CD8+ T-cell response, which was improved in combination with checkpoint immunotherapy. These results suggest a novel role for Axl in suppressing antigen presentation through MHCI, and enhancing cytokine release, which promotes a suppressive myeloid microenvironment.

The hypoxia-inducible factor (Hif)-1α (Hif-1α) and Hif-2α (Epas1) have a critical role in both normal development and cancer. von Hippel Lindau (Vhl) protein, encoded by a tumor suppressor gene, is an E3 ubiquitin ligase that targets Hif-1α and Epas1 to the proteasome for degradation. To better understand the role of Vhl in the biology of mesenchymal cells, we analyzed mutant mice lacking Vhl in mesenchymal progenitors that give rise to the soft tissues that form and surround synovial joints. Loss of Vhl in mesenchymal progenitors of the limb bud caused severe fibrosis of the synovial joints and formation of aggressive masses with histologic features of mesenchymal tumors. Hif-1α and its downstream target connective tissue growth factor were necessary for the development of these tumors, which conversely still developed in the absence of Epas1, but at lower frequency. Human tumors of the soft tissue are a very complex and heterogeneous group of neoplasias. Our novel findings in genetically altered mice suggest that activation of the HIF signaling pathway could be an important pathogenetic event in the development and progression of at least a subset of these tumors.

The induction of hypoxia-inducible factors (HIFs) is essential for the adaptation of tumor cells to a low-oxygen environment. We found that the expression of the apoptosis inhibitor ARC (apoptosis repressor with a CARD domain) was induced by hypoxia in a variety of cancer cell types, and its induction is primarily HIF1 dependent. Chromatin immunoprecipitation (ChIP) and reporter assays also indicate that the ARC gene is regulated by direct binding of HIF1 to a hypoxia response element (HRE) located at bp -190 upstream of the transcription start site. HIFs play an essential role in the pathogenesis of renal cell carcinoma (RCC) under normoxic conditions, through the loss of the Von Hippel-Lindau gene (VHL). Accordingly, our results show that ARC is not expressed in normal renal tissue but is highly expressed in 65% of RCC tumors, which also express high levels of carbonic anhydrase IX (CAIX), a HIF1-dependent protein. Compared to controls, ARC-deficient RCCs exhibited decreased colony formation and increased apoptosis in vitro. In addition, loss of ARC resulted in a dramatic reduction of RCC tumor growth in SCID mice in vivo. Thus, HIF-mediated increased expression of ARC in RCC can explain how loss of VHL can promote survival early in tumor formation.