Hypoxic adaptation of mitochondria and its impact on tumor cell function.

Mitochondria are the major sink for oxygen in the cell, consuming it during ATP production. Therefore, when environmental oxygen levels drop in the tumor, significant adaptation is required. Mitochondrial activity is also a major producer of biosynthetic precursors and a regulator of cellular oxidative and reductive balance. Because of the complex biochemistry, mitochondrial adaptation to hypoxia occurs through multiple mechanisms and has significant impact on other cellular processes such as macromolecule synthesis and gene regulation. In tumor hypoxia, mitochondria shift their location in the cell and accelerate the fission and quality control pathways. Hypoxic mitochondria also undergo significant changes to fundamental metabolic pathways of carbon metabolism and electron transport. These metabolic changes further impact the nuclear epigenome because mitochondrial metabolites are used as enzymatic substrates for modifying chromatin. This coordinated response delivers physiological flexibility and increased tumor cell robustness during the environmental stress of low oxygen.

Nutrient scavenging-fueled growth in pancreatic cancer depends on caveolae-mediated endocytosis under nutrient-deprived conditions.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by its nutrient-scavenging ability, crucial for tumor progression. Here, we investigated the roles of caveolae-mediated endocytosis (CME) in PDAC progression. Analysis of patient data across diverse datasets revealed a strong association of high caveolin-1 (Cav-1) expression with higher histologic grade, the most aggressive PDAC molecular subtypes, and worse clinical outcomes. Cav-1 loss markedly promoted longer overall and tumor-free survival in a genetically engineered mouse model. Cav-1-deficient tumor cell lines exhibited significantly reduced proliferation, particularly under low nutrient conditions. Supplementing cells with albumin rescued the growth of Cav-1-proficient PDAC cells, but not in Cav-1-deficient PDAC cells under low glutamine conditions. In addition, Cav-1 depletion led to significant metabolic defects, including decreased glycolytic and mitochondrial metabolism, and downstream protein translation signaling pathways. These findings highlight the crucial role of Cav-1 and CME in fueling pancreatic tumorigenesis, sustaining tumor growth, and promoting survival through nutrient scavenging.

A Bioinformatics Tool for Identifying Intratumoral Microbes from the ORIEN Dataset.

Evidence supports significant interactions among microbes, immune cells, and tumor cells in at least 10%-20% of human cancers, emphasizing the importance of further investigating these complex relationships. However, the implications and significance of tumor-related microbes remain largely unknown. Studies have demonstrated the critical roles of host microbes in cancer prevention and treatment responses. Understanding interactions between host microbes and cancer can drive cancer diagnosis and microbial therapeutics (bugs as drugs). Computational identification of cancer-specific microbes and their associations is still challenging due to the high dimensionality and high sparsity of intratumoral microbiome data, which requires large datasets containing sufficient event observations to identify relationships, and the interactions within microbial communities, the heterogeneity in microbial composition, and other confounding effects that can lead to spurious associations. To solve these issues, we present a bioinformatics tool, microbial graph attention (MEGA), to identify the microbes most strongly associated with 12 cancer types. We demonstrate its utility on a dataset from a consortium of nine cancer centers in the Oncology Research Information Exchange Network. This package has three unique features: species-sample relations are represented in a heterogeneous graph and learned by a graph attention network; it incorporates metabolic and phylogenetic information to reflect intricate relationships within microbial communities; and it provides multiple functionalities for association interpretations and visualizations. We analyzed 2,704 tumor RNA sequencing samples and MEGA interpreted the tissue-resident microbial signatures of each of 12 cancer types. MEGA can effectively identify cancer-associated microbial signatures and refine their interactions with tumors. SIGNIFICANCE: Studying the tumor microbiome in high-throughput sequencing data is challenging because of the extremely sparse data matrices, heterogeneity, and high likelihood of contamination. We present a new deep learning tool, MEGA, to refine the organisms that interact with tumors.

Exogenous Sequences in Tumors and Immune Cells (Exotic): A Tool for Estimating the Microbe Abundances in Tumor RNA-seq Data.

The microbiome affects cancer, from carcinogenesis to response to treatments. New evidence suggests that microbes are also present in many tumors, though the scope of how they affect tumor biology and clinical outcomes is in its early stages. A broad survey of tumor microbiome samples across several independent datasets is needed to identify robust correlations for follow-up testing. We created a tool called {exotic} for "exogenous sequences in tumors and immune cells" to carefully identify the tumor microbiome within RNA sequencing (RNA-seq) datasets. We applied it to samples collected through the Oncology Research Information Exchange Network (ORIEN) and The Cancer Genome Atlas. We showed how the processing removes contaminants and batch effects to yield microbe abundances consistent with non-high-throughput sequencing-based approaches and DNA-amplicon-based measurements of a subset of the same tumors. We sought to establish clinical relevance by correlating the microbe abundances with various clinical and tumor measurements, such as age and tumor hypoxia. This process leveraged the two datasets and raised up only the concordant (significant and in the same direction) associations. We observed associations with survival and clinical variables that are cancer specific and relatively few associations with immune composition. Finally, we explored potential mechanisms by which microbes and tumors may interact using a network-based approach. Alistipes, a common gut commensal, showed the highest network degree centrality and was associated with genes related to metabolism and inflammation. The {exotic} tool can support the discovery of microbes in tumors in a way that leverages the many existing and growing RNA-seq datasets. SIGNIFICANCE: The intrinsic tumor microbiome holds great potential for its ability to predict various aspects of cancer biology and as a target for rational manipulation. Here, we describe a tool to quantify microbes from within tumor RNA-seq and apply it to two independent datasets. We show new associations with clinical variables that justify biomarker uses and more experimentation into the mechanisms by which tumor microbiomes affect cancer outcomes.

The tumor microbiome as a predictor of outcomes in patients with metastatic melanoma treated with immune checkpoint inhibitors.

Emerging evidence supports the important role of the tumor microbiome in oncogenesis, cancer immune phenotype, cancer progression, and treatment outcomes in many malignancies. In this study, we investigated the metastatic melanoma tumor microbiome and potential roles in association with clinical outcomes, such as survival, in patients with metastatic disease treated with immune checkpoint inhibitors (ICIs). Baseline tumor samples were collected from 71 patients with metastatic melanoma before treatment with ICIs. Bulk RNA-seq was conducted on the formalin-fixed paraffin-embedded (FFPE) tumor samples. Durable clinical benefit (primary clinical endpoint) following ICIs was defined as overall survival ≥24 months and no change to the primary drug regimen (responders). We processed RNA-seq reads to carefully identify exogenous sequences using the {exotic} tool. The 71 patients with metastatic melanoma ranged in age from 24 to 83 years, 59% were male, and 55% survived >24 months following the initiation of ICI treatment. Exogenous taxa were identified in the tumor RNA-seq, including bacteria, fungi, and viruses. We found differences in gene expression and microbe abundances in immunotherapy responsive versus non-responsive tumors. Responders showed significant enrichment of several microbes including Fusobacterium nucleatum, and non-responders showed enrichment of fungi, as well as several bacteria. These microbes correlated with immune-related gene expression signatures. Finally, we found that models for predicting prolonged survival with immunotherapy using both microbe abundances and gene expression outperformed models using either dataset alone. Our findings warrant further investigation and potentially support therapeutic strategies to modify the tumor microbiome in order to improve treatment outcomes with ICIs.

A bioinformatics tool for identifying intratumoral microbes from the ORIEN dataset.

Evidence supports significant interactions among microbes, immune cells, and tumor cells in at least 10-20% of human cancers, emphasizing the importance of further investigating these complex relationships. However, the implications and significance of tumor-related microbes remain largely unknown. Studies have demonstrated the critical roles of host microbes in cancer prevention and treatment responses. Understanding interactions between host microbes and cancer can drive cancer diagnosis and microbial therapeutics (bugs as drugs). Computational identification of cancer-specific microbes and their associations is still challenging due to the high dimensionality and high sparsity of intratumoral microbiome data, which requires large datasets containing sufficient event observations to identify relationships, and the interactions within microbial communities, the heterogeneity in microbial composition, and other confounding effects that can lead to spurious associations. To solve these issues, we present a bioinformatics tool, MEGA, to identify the microbes most strongly associated with 12 cancer types. We demonstrate its utility on a dataset from a consortium of 9 cancer centers in the Oncology Research Information Exchange Network (ORIEN). This package has 3 unique features: species-sample relations are represented in a heterogeneous graph and learned by a graph attention network; it incorporates metabolic and phylogenetic information to reflect intricate relationships within microbial communities; and it provides multiple functionalities for association interpretations and visualizations. We analyzed 2704 tumor RNA-seq samples and MEGA interpreted the tissue-resident microbial signatures of each of 12 cancer types. MEGA can effectively identify cancer-associated microbial signatures and refine their interactions with tumors.

Unanticipated metabolic plasticity in response to chronic hypoxia.

In this issue of Cell Metabolism, Midha et al. investigate the metabolic changes in mice after exposure to reduced oxygen tension for an acute or chronic duration. Their organ-specific findings may help explain physiological observations in humans living at high altitude but raise additional questions concerning pathological hypoxia after vascular damage or in cancer.

Association between Tumor Microbiome and Hypoxia across Anatomic Subsites of Head and Neck Cancers.

Purpose/Objective(s): Microbiome has been shown to affect tumorigenesis by promoting inflammation. However, the association between the upper aerodigestive microbiome and head and neck squamous cell carcinoma (HNSCC) is not well established. Hypoxia is a modifiable factor associated with poor radiation response. Our study analyzed the HNSCC tumor samples from The Cancer Genome Atlas (TCGA) to investigate the relationship between different HNSCC tumor subsites, hypoxia, and local tumor microbiome composition. Results: A total of 357 patients were included [Oral cavity (OC) = 226, Oropharynx (OPx) = 53, and Larynx/Hypopharynx (LHPx) = 78], of which 12.8%, 71.7%, and 10.3%, respectively, were HPV positive. The mean (SD) hypoxia scores were 30.18 (11.10), 24.31 (14.13), and 29.53 (12.61) in OC, OPx, and LHPx tumors, respectively, with higher values indicating greater hypoxia. The hypoxia score was significantly higher for OC tumors compared to OPx (p = 0.044) and LHPx (p = 0.002). There was no significant correlation between hypoxia and HPV status. Pseudomonas sp. in OC, Actinomyces sp. and Sulfurimonas sp. in OPx, and Filifactor, Pseudomonas and Actinomyces sp. in LHPx had the strongest association with the hypoxia score. Materials/Methods: Tumor RNAseq samples from TCGA were processed, and the R package "tmesig" was used to calculate gene expression signature, including the Buffa hypoxia (BH) score, a validated hypoxia signature using 52 hypoxia-regulated genes. Microbe relative abundances were modeled with primary tumor location and a high vs. low tertile BH score applying a gamma-distributed generalized linear regression using the "stats" package in R, with adjusted p-value < 0.05 considered significant. Conclusions: In our study, oral cavity tumors were found to be more hypoxic compared to other head and neck subsites, which could potentially contribute to their radiation resistance. For each subsite, distinct microbial populations were over-represented in hypoxic tumors in a subsite-specific manner. Further studies focusing on an association between microbiome, hypoxia, and patient outcomes are warranted.

Aldehyde dehydrogenase 3A1 deficiency leads to mitochondrial dysfunction and impacts salivary gland stem cell phenotype.

Adult salivary stem/progenitor cells (SSPC) have an intrinsic property to self-renew in order to maintain tissue architecture and homeostasis. Adult salivary glands have been documented to harbor SSPC, which have been shown to play a vital role in the regeneration of the glandular structures postradiation damage. We have previously demonstrated that activation of aldehyde dehydrogenase 3A1 (ALDH3A1) after radiation reduced aldehyde accumulation in SSPC, leading to less apoptosis and improved salivary function. We subsequently found that sustained pharmacological ALDH3A1 activation is critical to enhance regeneration of murine submandibular gland after radiation damage. Further investigation shows that ALDH3A1 function is crucial for SSPC self-renewal and survival even in the absence of radiation stress. Salivary glands from Aldh3a1 -/- mice have fewer acinar structures than wildtype mice. ALDH3A1 deletion or pharmacological inhibition in SSPC leads to a decrease in mitochondrial DNA copy number, lower expression of mitochondrial specific genes and proteins, structural abnormalities, lower membrane potential, and reduced cellular respiration. Loss or inhibition of ALDH3A1 also elevates ROS levels, depletes glutathione pool, and accumulates ALDH3A1 substrate 4-hydroxynonenal (4-HNE, a lipid peroxidation product), leading to decreased survival of murine SSPC that can be rescued by treatment with 4-HNE specific carbonyl scavengers. Our data indicate that ALDH3A1 activity protects mitochondrial function and is important for the regeneration activity of SSPC. This knowledge will help to guide our translational strategy of applying ALDH3A1 activators in the clinic to prevent radiation-related hyposalivation in head and neck cancer patients.

Slit2-Mediated Metabolic Reprogramming in Bone Marrow-Derived Macrophages Enhances Antitumor Immunity.

Slit2 exerts antitumor effects in various cancers; however, the underlying mechanism, especially its role in regulating the immune, especially in the bone marrow niche, system is still unknown. Elucidating the behavior of macrophages in tumor progression can potentially improve immunotherapy. Using a spontaneous mammary tumor virus promoter-polyoma middle T antigen (PyMT) breast cancer mouse model, we observed that Slit2 increased the abundance of antitumor M1 macrophage in the bone marrow upon differentiation in vitro. Moreover, myeloablated PyMT mice injected with Slit2-treated bone marrow allografts showed a marked reduction in tumor growth, with enhanced recruitment of M1 macrophage in their tumor stroma. Mechanistic studies revealed that Slit2 significantly enhanced glycolysis and reduced fatty acid oxidation in bone marrow-derived macrophages (BMDMs). Slit2 treatment also altered mitochondrial respiration metabolites in macrophages isolated from healthy human blood that were treated with plasma from breast cancer patients. Overall, this study, for the first time, shows that Slit2 increases BMDM polarization toward antitumor phenotype by modulating immune-metabolism. Furthermore, this study provides evidence that soluble Slit2 could be developed as novel therapeutic strategy to enhance antitumor immune response.

Dynamic regulation of mitochondrial pyruvate metabolism is necessary for orthotopic pancreatic tumor growth.

BACKGROUND: Pyruvate dehydrogenase complex (PDC) plays a central role in carbohydrate metabolism, linking cytoplasmic glycolysis to the mitochondrial tricarboxylic acid (TCA) cycle. PDC is a conserved E1-E2-E3 dehydrogenase with a PDHA1 and PDHB heterotetramer functioning as the E1 subunit. PDHA1 contains three serine residues that can be reversibly phosphorylated by a dedicated family of four inhibitory pyruvate dehydrogenase kinases (PDHK1-4) and two reactivating phosphatases (PDP1, 2). Hypoxia induces the expression of PDHK1 and PDHK3 and hyperphosphorylates PDHA1. The role of PDC in metabolic reprogramming and tumor progression appears to be for the integration of oncogenic and environmental signals which supports tumor growth. METHODS: To isolate the function of the serine-dependent regulation of PDC, we engineered MiaPaca2 cells to express PDHA1 protein with either intact serines at positions 232, 293, and 300 or all the combinations of non-phosphorylatable alanine substitution mutations. These lines were compared in vitro for biochemical response to hypoxia by western blot, metabolic activity by biochemical assay and Seahorse XF flux analysis, and growth in media with reduced exogenous metabolites. The lines were also tested for growth in vivo after orthotopic injection into the pancreata of immune-deficient mice. RESULTS: In this family of cells with non-phosphorylatable PDHA1, we found reduced hypoxic phosphorylation of PDHA1, decreased PDH enzymatic activity in normoxia and hypoxia, decreased mitochondrial function by Seahorse flux assay, reduced in vitro growth of cells in media depleted of lipids, and reduced growth of tumors after orthotopic transplantation of cells into the pancreata of immune-deficient mice. CONCLUSIONS: We found that any substitution of alanine for serine at regulatory sites generated a hypomorphic PDC. However, the reduced PDC activity was insensitive to further reduction in hypoxia. These cells had a very modest reduction of growth in vitro, but failed to grow as tumors indicating that dynamic PDC adaptation to microenvironmental conditions is necessary to support pancreatic cancer growth in vivo.

Effect of glucose concentration and cryopreservation on mitochondrial functions of bull spermatozoa and relationship with sire conception rate.

Mitochondrial function is essential for sperm viability, not only from a sperm metabolism perspective, but also for improvement of sperm storage in liquid and frozen states. Bull sperm have notable metabolic variability with energy production for motility and subsequently for fertilizing capacity resulting from both glycolysis and oxidative phosphorylation. The objective of this study was to determine mitochondrial function of sperm using high-throughput Seahorse Analyzer technology in fresh semen and subsequent to freezing-thawing when there was incubation in media commonly used for sperm storage (relatively large glucose concentration) and female tract (relatively small glucose concentration). Additionally, there were determinations whether there were differences in values for fertility variables by regressing sire conception rate on values for mitochondrial variables when there was evaluation of semen from bulls with varying fertility. Media with larger concentrations of glucose inhibited mitochondrial function in fresh sperm, as indicated by less maximal oxygen consumption, spare respiratory capacity and coupling efficiency when compared to sperm in the media containing less glucose. Furthermore, there was greater (P <  0.05) mitochondrial function in cryopreserved-thawed compared to fresh samples with there being no effect of incubation media. These results indicate that mitochondrial damage from cryopreservation cannot be simply overcome post-thawing with glucose supplementation of bull semen incubation media. The increase in mitochondrial function is likely due to "non-productive" oxygen consumption to maintain the mitochondrial proton gradient. Furthermore, there was a negative association of mitochondrial proton leakage with sire conception rate indicating this could be a potential biomarker of bull fertility.

Pharmacological Regulation of Tumor Hypoxia in Model Murine Tumors and Spontaneous Canine Tumors.

BACKGROUND: Hypoxia is found in many solid tumors and is associated with increased disease aggressiveness and resistance to therapy. Reducing oxygen demand by targeting mitochondrial oxidative metabolism is an emerging concept in translational cancer research aimed at reducing hypoxia. We have shown that the U.S. Food and Drug Administration (FDA)-approved drug papaverine and its novel derivative SMV-32 are potent mitochondrial complex I inhibitors. METHODS: We used a dynamic in vivo luciferase reporter system, pODD-Luc, to evaluate the impact of pharmacological manipulation of mitochondrial metabolism on the levels of tumor hypoxia in transplanted mouse tumors. We also imaged canine patients with blood oxygen level-dependent (BOLD) MRI at baseline and one hour after a dose of 1 or 2 mg/kg papaverine. RESULTS: We showed that the pharmacological suppression of mitochondrial oxygen consumption (OCR) in tumor-bearing mice increases tumor oxygenation, while the stimulation of mitochondrial OCR decreases tumor oxygenation. In parallel experiments in a small series of spontaneous canine sarcomas treated at The Ohio State University (OSU) Veterinary Medical Center, we observed a significant increase in BOLD signals indicative of an increase in tumor oxygenation of up to 10-50 mm HgO2. CONCLUSION: In both transplanted murine tumors and spontaneous canine tumors we found that decreasing mitochondrial metabolism can decrease tumor hypoxia, potentially offering a therapeutic advantage.

Lipid droplet storage promotes murine pancreatic tumor growth.

Hypoxia Inducible Lipid Droplet Associated (HILPDA) is frequently overexpressed in tumors and promotes neutral lipid storage. The impact of Hilpda on pancreatic ductal adenocarcinoma (PDAC) tumor growth is not known. In order to evaluate Hilpda­dependent lipid storage mechanisms, expression of Hilpda in murine pancreatic cells (KPC) was genetically manipulated. Lipid droplet (LD) abundance and triglyceride content in vitro were measured, and model tumor growth in nu/nu mice was determined. The results showed that excess lipid supply increased triglyceride storage and LD formation in KPC cells in a HILPDA­dependent manner. Contrary to published results, inhibition of Adipose Triglyceride Lipase (ATGL) did not ameliorate the triglyceride abundance differences between Hilpda WT and KO cells. Hilpda ablation significantly decreased the growth rate of model tumors in immunocompromised mice. In conclusion, Hilpda is a positive regulator of triglyceride storage and lipid droplet formation in murine pancreatic cancer cells in vitro and lipid accumulation and tumor growth in vivo. Our data suggest that deregulated ATGL is not responsible for the absence of LDs in KO cells in this context.

CA IX Stabilizes Intracellular pH to Maintain Metabolic Reprogramming and Proliferation in Hypoxia.

Tumor hypoxia represents a severe microenvironmental stress that is frequently associated with acidosis. Cancer cells respond to these stresses with changes in gene expression that promote survival at least in part through pH regulation and metabolic reprogramming. Hypoxia-induced carbonic anhydrase IX (CA IX) plays a critical adaptive role in response to hypoxic and acidic environments by catalytically hydrating extracellular CO2 to produce bicarbonate for buffering intracellular pH (pHi). We used proteome-wide profiling to study the cellular response to transient CA IX knockdown in hypoxia and found a decrease in the levels of key glycolytic enzymes and lactate dehydrogenase A (LDHA). Interestingly, the activity of LDH was also decreased as demonstrated by native in-gel activity assay. These changes led to a significant reduction in glycolytic flux and extracellular lactate levels in cancer cells in vitro, contributing to a decrease in proliferation. Interestingly, addition of the alternative LDH substrate alpha-ketobutyrate restored LDHA activity, extracellular acidification, pHi, and cellular proliferation. These results indicate that in the absence of CA IX, reduction of pHi disrupts LDHA activity and hinders the cellular capacity to regenerate NAD+ and secrete protons to the extracellular space. Hypoxia-induced CA IX therefore mediates adaptation to microenvironmental hypoxia and acidosis directly, by enzymatically converting extracellular CO2 to bicarbonate, and indirectly, by maintaining glycolysis-permissive intracellular milieu.

HILPDA Regulates Lipid Metabolism, Lipid Droplet Abundance, and Response to Microenvironmental Stress in Solid Tumors.

Accumulation of lipid droplets has been observed in an increasing range of tumors. However, the molecular determinants of this phenotype and the impact of the tumor microenvironment on lipid droplet dynamics are not well defined. The hypoxia-inducible and lipid droplet associated protein HILPDA is known to regulate lipid storage and physiologic responses to feeding conditions in mice, and was recently shown to promote hypoxic lipid droplet formation through inhibition of the rate-limiting lipase adipose triglyceride lipase (ATGL). Here, we identify fatty acid loading and nutrient deprivation-induced autophagy as stimuli of HILPDA-dependent lipid droplet growth. Using mouse embryonic fibroblasts and human tumor cells, we found that genetic ablation of HILPDA compromised hypoxia-fatty acid- and starvation-induced lipid droplet formation and triglyceride storage. Nutrient deprivation upregulated HILPDA protein posttranscriptionally by a mechanism requiring autophagic flux and lipid droplet turnover, independent of HIF1 transactivation. Mechanistically, loss of HILPDA led to elevated lipolysis, which could be corrected by inhibition of ATGL. Lipidomic analysis revealed not only quantitative but also qualitative differences in the glycerolipid and phospholipid profile of HILPDA wild-type and knockout cells, indicating additional HILPDA functions affecting lipid metabolism. Deletion studies of HILPDA mutants identified the N-terminal hydrophobic domain as sufficient for targeting to lipid droplets and restoration of triglyceride storage. In vivo, HILPDA-ablated cells showed decreased intratumoral triglyceride levels and impaired xenograft tumor growth associated with elevated levels of apoptosis. IMPLICATIONS: Tumor microenvironmental stresses induce changes in lipid droplet dynamics via HILPDA. Regulation of triglyceride hydrolysis is crucial for cell homeostasis and tumor growth.

Papaverine and its derivatives radiosensitize solid tumors by inhibiting mitochondrial metabolism.

Tumor hypoxia reduces the effectiveness of radiation therapy by limiting the biologically effective dose. An acute increase in tumor oxygenation before radiation treatment should therefore significantly improve the tumor cell kill after radiation. Efforts to increase oxygen delivery to the tumor have not shown positive clinical results. Here we show that targeting mitochondrial respiration results in a significant reduction of the tumor cells' demand for oxygen, leading to increased tumor oxygenation and radiation response. We identified an activity of the FDA-approved drug papaverine as an inhibitor of mitochondrial complex I. We also provide genetic evidence that papaverine's complex I inhibition is directly responsible for increased oxygenation and enhanced radiation response. Furthermore, we describe derivatives of papaverine that have the potential to become clinical radiosensitizers with potentially fewer side effects. Importantly, this radiosensitizing strategy will not sensitize well-oxygenated normal tissue, thereby increasing the therapeutic index of radiotherapy.

A Human Genome-Wide RNAi Screen Reveals Diverse Modulators that Mediate IRE1α-XBP1 Activation.

Activation of the unfolded protein response (UPR) signaling pathways is linked to multiple human diseases, including cancer. The inositol-requiring kinase 1α (IRE1α)-X-box binding protein 1 (XBP1) pathway is the most evolutionarily conserved of the three major signaling branches of the UPR. Here, we performed a genome-wide siRNA screen to obtain a systematic assessment of genes integrated in the IRE1α-XBP1 axis. We monitored the expression of an XBP1-luciferase chimeric protein in which luciferase was fused in-frame with the spliced (active) form of XBP1. Using cells expressing this reporter construct, we identified 162 genes for which siRNA inhibition resulted in alteration in XBP1 splicing. These genes express diverse types of proteins modulating a wide range of cellular processes. Pathway analysis identified a set of genes implicated in the pathogenesis of breast cancer. Several genes, including BCL10, GCLM, and IGF1R, correlated with worse relapse-free survival (RFS) in an analysis of patients with triple-negative breast cancer (TNBC). However, in this cohort of 1,908 patients, only high GCLM expression correlated with worse RFS in both TNBC and non-TNBC patients. Altogether, our study revealed unidentified roles of novel pathways regulating the UPR, and these findings may serve as a paradigm for exploring novel therapeutic opportunities based on modulating the UPR.Implications: Genome-wide RNAi screen identifies novel genes/pathways that modulate IRE1α-XBP1 signaling in human tumor cells and leads to the development of improved therapeutic approaches targeting the UPR.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/16/5/745/F1.large.jpg Mol Cancer Res; 16(5); 745-53. ©2018 AACR.

Urinary miRNAs as Biomarkers for Noninvasive Evaluation of Radiation-Induced Renal Tubular Injury.

Radiation nephropathy is one of the common late effects in cancer survivors who received radiotherapy as well as in victims of radiation accidents. The clinical manifestations of radiation nephropathy occur months after exposure. To date, there are no known early biomarkers to predict the future development of radiation nephropathy. This study focuses on the development of urinary biomarkers providing readout of acute responses in renal tubular epithelial cells. An amplification-free hybridization-based nCounter assay was used to detect changes in mouse urinary miRNAs after irradiation. After a single LD50 of total-body irradiation (TBI) or clinically relevant fractionated doses (2 Gy twice daily for 3 days), changes in urinary levels of microRNAs followed either an early pattern, peaking at 6-8 h postirradiation and gradually declining, or later pattern, peaking from 24 h to 7 days. Of 600 miRNAs compared, 12 urinary miRNAs showed the acute response and seven showed the late response, common to both irradiation protocols. miR-1224 and miR-21 were of particular interest, since they were the most robust acute and late responders, respectively. The early responding miR-1224 also exhibited good dose response after 2, 4, 6 and 8 Gy TBI, indicating its potential use as a biomarker for radiation exposure. In situ hybridization of irradiated mouse kidney sections and cultured mouse primary renal tubular cells confirmed the tubular origin of miR-1224. A significant upregulation in hsa-miR-1224-3p expression was also observed in human proximal renal tubular cells after irradiation. Consistent with mouse urine data, a similar expression pattern of hsa-miR-1224-3p and hsa-miR-21 were observed in urine samples collected from human leukemia patients preconditioned with TBI. This proof-of-concept study shows the potential translational utility of urinary miRNA biomarkers for radiation damage in renal tubules with possible prediction of late effects.

Stress-responsive HILPDA is necessary for thermoregulation during fasting.

Hypoxia-inducible lipid droplet-associated protein (HILPDA) has been shown to localize to lipid droplets in nutrient-responsive cell types such as hepatocytes and adipocytes. However, its role in the control of whole-body homeostasis is not known. We sought to measure cell-intrinsic and systemic stress responses in a mouse strain harboring whole-body Hilpda deficiency. We generated a genetically engineered mouse model of whole-body HILPDA deficiency by replacing the coding Hilpda exon with luciferase. We subjected the knockout animals to environmental stresses and measured whole-animal metabolic and behavioral parameters. Brown adipocyte precursors were isolated and differentiated in vitro to quantify the impact of HILPDA ablation in lipid storage and mobilization in these cells. HILPDA-knockout animals are viable and fertile, but show reduced ambulatory activity and oxygen consumption at regular housing conditions. Acclimatization at thermoneutral conditions abolished the phenotypic differences observed at 22°C. When fasted, HILPDA KO mice are unable to maintain body temperature and become hypothermic at 22°C, without apparent abnormalities in blood chemistry parameters or tissue triglyceride content. HILPDA expression was upregulated during adipocyte differentiation and activation in vitro; however, it was not required for lipid droplet formation in brown adipocytes. We conclude that HILPDA is necessary for efficient fuel utilization suggesting a homeostatic role for Hilpda in sub-optimal environments.