Hypoxia-Inducible Factor-1: A Critical Player in the Survival Strategy of Stressed Cells.

HIF-1 activation has been well known as an adaptive strategy to hypoxia. Recently it became clear that hypoxia was often accompanied by insufficient supply of glucose or amino acids as a common result of poor circulation that frequently occurs in solid tumors and ischemic lesions, creating a mixed nutrient insufficiency. In response to nutrient insufficiency, stressed cells elicit survival strategies including activation of AMPK and HIF-1 to cope with the stress. Particularly, in solid tumors, HIF-1 promotes cell survival and migration, stimulates angiogenesis, and induces resistance to radiation and chemotherapy. Interestingly, radiation and some chemotherapeutics are reported to trigger the activation of AMPK. Here we discuss the recent advances that may potentially link the stress responsive mechanisms including AMPK activation, ATF4 activation and the enhancement of Hsp70/Hsp90 function to HIF-1 activation. Potential implication and application of the stress-facilitated HIF-1 activation in solid tumors and ischemic disorders will be discussed. A better understanding of HIF-1 activation in cells exposed to stresses is expected to facilitate the design of therapeutic approaches that specifically modulate cell survival strategy.

Cathepsin L Mediates the Degradation of Novel APP C-Terminal Fragments.

Alzheimer's disease (AD) is characterized by the deposition of amyloid ß (Aß), a peptide generated from proteolytic processing of its precursor, amyloid precursor protein (APP). Canonical APP proteolysis occurs via α-, ß-, and γ-secretases. APP is also actively degraded by protein degradation systems. By pharmacologically inhibiting protein degradation with ALLN, we observed an accumulation of several novel APP C-terminal fragments (CTFs). The two major novel CTFs migrated around 15 and 25 kDa and can be observed across multiple cell types. The process was independent of cytotoxicity or protein synthesis. We further determine that the accumulation of the novel CTFs is not mediated by proteasome or calpain inhibition, but by cathepsin L inhibition. Moreover, these novel CTFs are not generated by an increased amount of BACE. Here, we name the CTF of 25 kDa as η-CTF (eta-CTF). Our data suggest that under physiological conditions, a subset of APP undergoes alternative processing and the intermediate products, the 15 kDa CTFs, and the η-CTFs aret rapidly degraded and/or processed via the protein degradation machinery, specifically, cathepsin L.

ErbB2 activation upregulates glutaminase 1 expression which promotes breast cancer cell proliferation.

Active glutamine utilization is critical for tumor cell proliferation. Glutaminolysis represents the first and rate-limiting step of glutamine utilization and is catalyzed by glutaminase (GLS). Activation of ErbB2 is one of the major causes of breast cancers, the second most common cause of death for women in many countries. However, it remains unclear whether ErbB2 signaling affects glutaminase expression in breast cancer cells. In this study, we show that MCF10A-NeuT cell line has higher GLS1 expression at both mRNA and protein levels than its parental line MCF10A, and knockdown of ErbB2 decreases GLS1 expression in MCF10A-NeuT cells. We further show that in these cells, ErbB2-mediated upregulation of GLS1 is not correlated to c-Myc expression. Moreover, activation of neither PI3K-Akt nor MAPK pathway is sufficient to upregulate GLS1 expression. Interestingly, inhibition of NF-κB blocks ErbB2-stimulated GLS1 expression, whereas stimulation of NF-κB is sufficient to enhance GLS1 levels in MCF10A cells, suggesting a PI3K-Akt-independent activation of NF-κB upregulates GLS1 in ErbB2-positive breast cancer cells. Finally, knockdown or inhibition of GLS1 significantly decreased the proliferation of breast cancer cells with high GLS1 levels. Taken together, our data indicate that ErbB2 activation promotes GLS1 expression via a PI3K-Akt-independent NF-κB pathway in breast cancer cells, identifying another oncogenic signaling pathway which stimulates GLS1 expression, and thus promoting glutamine utilization in cancer cells. These findings, if validated by in vivo model, may facilitate the identification of novel biochemical targets for cancer prevention and therapy.

Stanniocalcin 2 governs cancer cell adaptation to nutrient insufficiency through alleviation of oxidative stress.

Solid tumours often endure nutrient insufficiency during progression. How tumour cells adapt to temporal and spatial nutrient insufficiency remains unclear. We previously identified STC2 as one of the most upregulated genes in cells exposed to nutrient insufficiency by transcriptome screening, indicating the potential of STC2 in cellular adaptation to nutrient insufficiency. However, the molecular mechanisms underlying STC2 induction by nutrient insufficiency and subsequent adaptation remain elusive. Here, we report that STC2 protein is dramatically increased and secreted into the culture media by Gln-/Glc-deprivation. STC2 promoter contains cis-elements that are activated by ATF4 and p65/RelA, two transcription factors activated by a variety of cellular stress. Biologically, STC2 induction and secretion promote cell survival but attenuate cell proliferation during nutrient insufficiency, thus switching the priority of cancer cells from proliferation to survival. Loss of STC2 impairs tumour growth by inducing both apoptosis and necrosis in mouse xenografts. Mechanistically, under nutrient insufficient conditions, cells have increased levels of reactive oxygen species (ROS), and lack of STC2 further elevates ROS levels that lead to increased apoptosis. RNA-Seq analyses reveal STC2 induction suppresses the expression of monoamine oxidase B (MAOB), a mitochondrial membrane enzyme that produces ROS. Moreover, a negative correlation between STC2 and MAOB levels is also identified in human tumour samples. Importantly, the administration of recombinant STC2 to the culture media effectively suppresses MAOB expression as well as apoptosis, suggesting STC2 functions in an autocrine/paracrine manner. Taken together, our findings indicate that nutrient insufficiency induces STC2 expression, which in turn governs the adaptation of cancer cells to nutrient insufficiency through the maintenance of redox homeostasis, highlighting the potential of STC2 as a therapeutic target for cancer treatment.

Stanniocalcin 2 governs cancer cell adaptation to nutrient insufficiency through alleviation of oxidative stress.

Solid tumours often endure nutrient insufficiency during progression. How tumour cells adapt to temporal and spatial nutrient insufficiency remains unclear. We previously identified STC2 as one of the most upregulated genes in cells exposed to nutrient insufficiency by transcriptome screening, indicating the potential of STC2 in cellular adaptation to nutrient insufficiency. However, the molecular mechanisms underlying STC2 induction by nutrient insufficiency and subsequent adaptation remain elusive. Here, we report that STC2 protein is dramatically increased and secreted into the culture media by Gln-/Glc- deprivation. STC2 promoter contains cis-elements that are activated by ATF4 and p65/RelA, two transcription factors activated by a variety of cellular stress. Biologically, STC2 induction and secretion promote cell survival but attenuate cell proliferation during nutrient insufficiency, thus switching the priority of cancer cells from proliferation to survival. Loss of STC2 impairs tumour growth by inducing both apoptosis and necrosis in mouse xenografts. Mechanistically, under nutrient insufficient conditions, cells have increased levels of reactive oxygen species (ROS), and lack of STC2 further elevates ROS levels that lead to increased apoptosis. RNA-Seq analyses reveal STC2 induction suppresses the expression of monoamine oxidase B (MAOB), a mitochondrial membrane enzyme that produces ROS. Moreover, a negative correlation between STC2 and MAOB levels is also identified in human tumour samples. Importantly, the administration of recombinant STC2 to the culture media effectively suppresses MAOB expression as well as apoptosis, suggesting STC2 functions in an autocrine/paracrine manner. Taken together, our findings indicate that nutrient insufficiency induces STC2 expression, which in turn governs the adaptation of cancer cells to nutrient insufficiency through the maintenance of redox homoeostasis, highlighting the potential of STC2 as a therapeutic target for cancer treatment.

Two mutations impair the stability and function of ubiquitin-activating enzyme (E1).

Protein ubiquitination plays critical roles in the regulation of multiple cellular processes including cell proliferation, signal transduction, oncogenesis, and hypoxic response. TS20 is a Balb3T3-derived cell line in which ubiquitination is inhibited by restrictive temperature. While TS20 has been used to elucidate the degradation of many important proteins including p53, p27, HIF-1α, and ornithine decarboxylase, the molecular basis of its temperature sensitivity has not been fully determined. We cloned full-length E1 cDNA from TS20. Sequencing analysis revealed two point mutations (nt736G to A and nt2313G to C) that lead to substitution of aa189A to T and aa714W to C, respectively. Transient transfection assays revealed that mutant E1 was less stable than its wild-type counterpart, and restrictive temperature (39°C) accelerated its degradation. Under permissive temperature, reverting aa714C to W significantly improved E1 stability and activity. Under restrictive temperature, reverting of both substitutions was required to fully restore E1 stability. Similar results were observed when the mutants were expressed in non-TS20 cells, indicating the mutations are sufficient for its temperature sensitive degradation observed in TS20 cells. Functionally, reverting aa714C to W was sufficient to facilitate the monoubiquitination of H2A and to support TS20 growth at 39°C. It also significantly improved the ubiquitination-dependent disposal of HIF-1α. Our data conclusively demonstrate that mutations introgenic to UVBE1 cause E1 instability, which leads to deficiency of E1 function. Our data establish the molecular basis for unambiguous interpretation of experimental data based on TS20 cells, and provide new insight into the structural determinants of E1 stability.

Carbon source metabolism and its regulation in cancer cells.

Cancer cell proliferation and progression require sufficient supplies of nutrients including carbon sources, nitrogen sources, and molecular oxygen. Particularly, carbon sources and molecular oxygen are critical for the generation of ATP and building blocks, and for the maintenance of intracellular redox status. However, solid tumors frequently outgrow the blood supply, resulting in nutrient insufficiency. Accordingly, cancer cell metabolism shows aberrant biochemical features that are consequences of oncogenic signaling and adaptation. Those adaptive metabolism features, including the Warburg effect and addiction to glutamine, may form the biochemical basis for resistance to chemotherapy and radiation. A better understanding of the regulatory mechanisms that link the signaling pathways to adaptive metabolic reprogramming may identify novel biomarkers for drug development. In this review, we focus on the regulation of carbon source utilization at a cellular level, emphasizing its relevance to proliferative biosynthesis in cancer cells. We summarize the essential needs of proliferating cells and the metabolic features of glucose, lipids, and glutamine, and we review the roles of transcription regulators (i.e., HIF-1, c-Myc, and p53) and two major oncogenic signaling pathways (i.e., PI3K-Akt and MAPK) in regulating the utilization of carbon sources. Finally, the effects of glucose on cell proliferation and perspective from both biochemical and cellular angles are discussed.

Stanniocalcin 2 (STC2): a universal tumour biomarker and a potential therapeutical target.

Stanniocalcin 2 (STC2) is a glycoprotein which is expressed in a broad spectrum of tumour cells and tumour tissues derived from human breast, colorectum, stomach, esophagus, prostate, kidney, liver, bone, ovary, lung and so forth. The expression of STC2 is regulated at both transcriptional and post-transcriptional levels; particularly, STC2 is significantly stimulated under various stress conditions like ER stress, hypoxia and nutrient deprivation. Biologically, STC2 facilitates cells dealing with stress conditions and prevents apoptosis. Importantly, STC2 also promotes the development of acquired resistance to chemo- and radio- therapies. In addition, multiple groups have reported that STC2 overexpression promotes cell proliferation, migration and immune response. Therefore, the overexpression of STC2 is positively correlated with tumour growth, invasion, metastasis and patients' prognosis, highlighting its potential as a biomarker and a therapeutic target. This review focuses on discussing the regulation, biological functions and clinical importance of STC2 in human cancers. Future perspectives in this field will also be discussed.

Subgenomic particles in rAAV vectors result from DNA lesion/break and non-homologous end joining of vector genomes.

Recombinant adeno-associated virus (rAAV) vectors have been developed for therapeutic treatment of genetic diseases. Current rAAV vectors administered to affected individuals often contain vector DNA-related contaminants. Here we present a thorough molecular analysis of the configuration of non-standard AAV genomes generated during rAAV production using single-molecule sequencing. In addition to the sub-vector genomic-size particles containing incomplete AAV genomes, our results showed that rAAV preparations were contaminated with multiple categories of subgenomic particles with a snapback genome (SBG) configuration or a vector genome with deletions. Through CRISPR and nuclease-based modeling in tissue culture cells, we identified that a potential mechanism leading to formation of non-canonical genome particles occurred through non-homologous end joining of fragmented vector genomes caused by genome lesions or DNA breaks present in the host cells. The results of this study advance our understanding of AAV vectors and provide new clues for improving vector efficiency and safety profiles for use in human gene therapy.

Histone deacetylase inhibitors: the epigenetic therapeutics that repress hypoxia-inducible factors.

Histone deacetylase inhibitors (HDACIs) have been actively explored as a new generation of chemotherapeutics for cancers, generally known as epigenetic therapeutics. Recent findings indicate that several types of HDACIs repress angiogenesis, a process essential for tumor metabolism and progression. Accumulating evidence supports that this repression is mediated by disrupting the function of hypoxia-inducible factors (HIF-1, HIF-2, and collectively, HIF), which are the master regulators of angiogenesis and cellular adaptation to hypoxia. Since HIF also regulate glucose metabolism, cell survival, microenvironment remodeling, and other alterations commonly required for tumor progression, they are considered as novel targets for cancer chemotherapy. Though the precise biochemical mechanism underlying the HDACI-triggered repression of HIF function remains unclear, potential cellular factors that may link the inhibition of deacetylase activity to the repression of HIF function have been proposed. Here we review published data that inhibitors of type I/II HDACs repress HIF function by either reducing functional HIF-1α levels, or repressing HIF-α transactivation activity. In addition, underlying mechanisms and potential proteins involved in the repression will be discussed. A thorough understanding of HDACI-induced repression of HIF function may facilitate the development of future therapies to either repress or promote angiogenesis for cancer or chronic ischemic disorders, respectively.

Gender differences in UV-induced skin inflammation, skin carcinogenesis and systemic damage.

Ultraviolet (UV) radiation-induced chronic inflammation contributes to all stages of skin tumor development. In addition, gender plays an important role in inflammatory diseases or cancer. In this study, histopathology changes, hematology, oxidative stress and inflammatory response were used to evaluate sex differences in UV-induced chronic inflammation-associated cancer development. The results showed that the male and female mice had photoaging damage at the 9th week. However, skin tumors only appeared in male mice at 31st week. Furthermore, UV increased ROS production, p65, p-p65, IL-6 and TNF-α protein expressions in skin, and these factors elevated more in male mouse model. Hematology results showed that the parameters of blood systemic inflammation were changed in different degrees in model groups, while the pathological results showed inflammatory cell infiltration in the internal organs of both model groups in varying degrees. These results indicate that there are gender differences in UV-induced skin inflammation, carcinogenesis and systemic damage. Moreover, male mice are more sensitive to UV irradiation, which may be responsible to greater oxidative stress and inflammatory damage.

Effects of Thermally Induced Configuration Changes on rAAV Genome's Enzymatic Accessibility.

Physical titers for recombinant adeno-associated viral (rAAV) vectors are measured by quantifying viral genomes. It is generally perceived that AAV virions disassemble and release DNA upon thermal treatment. Here, we present data on enzymatic accessibility of rAAV genomes when AAV virions were subjected to thermal treatment. For rAAV vectors with a normal genome size (≤4.7 kb), thermal treatment at 75°C-99°C allowed only ∼10% of genomes to be detectable by quantitative real-time PCR. In contrast, greater than 70% of AAV genomes can be detected under similar conditions for AAV vectors with an oversized genome (≥5.0 kb). The permeability of virions, as measured by ethidium bromide (EB) staining, was enhanced by thermal stimulation. These results suggest that in rAAV virions with standard-sized genomes, the capsid and DNA are close enough in proximity for heat-induced "crosslinking," which results in inaccessibility of vector DNA to enzymatic reactions. In contrast, rAAV vectors with oversized genomes release their DNA readily upon thermal treatment. These findings suggested that the spatial arrangement of capsid protein and DNA in AAV virions is genome-size dependent. These results provide a foundation for future improvement of vector assays, design, and applications.

Histone deacetylase inhibitors induce VHL and ubiquitin-independent proteasomal degradation of hypoxia-inducible factor 1alpha.

Adaptation to hypoxic microenvironment is critical for tumor survival and metastatic spread. Hypoxia-inducible factor 1alpha (HIF-1alpha) plays a key role in this adaptation by stimulating the production of proangiogenic factors and inducing enzymes necessary for anaerobic metabolism. Histone deacetylase inhibitors (HDACIs) produce a marked inhibition of HIF-1alpha expression and are currently in clinical trials partly based on their potent antiangiogenic effects. Although it has been postulated that HDACIs affect HIF-1alpha expression by enhancing its interactions with VHL (von Hippel Lindau), thus promoting its ubiquitination and degradation, the actual mechanisms by which HDACIs decrease HIF-1alpha levels are not clear. Here, we present data indicating that HDACIs induce the proteasomal degradation of HIF-1alpha by a mechanism that is independent of VHL and p53 and does not require the ubiquitin system. This degradation pathway involves the enhanced interaction of HIF-1alpha with HSP70 and is secondary to a disruption of the HSP70/HSP90 axis function that appears mediated by the activity of HDAC-6.

Histone deacetylase inhibitors synergize p300 autoacetylation that regulates its transactivation activity and complex formation.

p300/cyclic AMP-responsive element binding protein-binding protein (CBP) are general coactivators for multiple transcription factors involved in various cellular processes. Several highly conserved domains of p300/CBP serve as interacting sites for transcription factors and regulatory proteins. Particularly, the intrinsic histone acetyltransferase (HAT) activity and transactivation domains (TAD) play essential roles for their coactivating function. Autoacetylation of p300/CBP is commonly observed in cell-free HAT assays and has been implicated in the regulation of their HAT activity. Here, we show that six lysine-rich regions in several highly conserved functional domains of p300 are targeted by p300HAT for acetylation in cell-free systems. We show that p300 is susceptible to acetylation in cultured tumor cells and that its acetylation status is affected by histone deacetylase inhibitor trichostatin A. We further show that either treatment with deacetylase inhibitors or coexpression of Gal4-p300HAT, which alone has no transactivation activity, stimulates the activity of the COOH-terminal TAD of p300 (p300C-TAD). We have defined the minimal p300C-TAD and show that it is sufficient to respond to deacetylase inhibitors and is a substrate for p300HAT. Finally, we show that acetylated p300 possesses enhanced ability to interact with p53. Taken together, our data suggest that acetylation regulates p300C-TAD and that acetylation of p300/CBP may contribute to the dynamic regulation of their complex formation with various interacting partners.

Compounds targeting class II histone deacetylases do not cause panHDACI-associated impairment of megakaryocyte differentiation.

Histone deacetylase inhibitors (HDACIs) have demonstrated effectiveness against lymphomas and myelomas in clinical practice. However, common to all currently approved broad-acting HDACIs (panHDACIs) is dose-limiting thrombocytopenia, which has prevented wider use in cancer therapy. Using CD34+ hematopoietic stem cells (HSCs), we show that megakaryocyte (MK) cell maturation and differentiation are impaired by panHDACIs, correlating to clinical thrombocytopenia. Importantly, we demonstrate that inhibitors of class II histone deacetylases (HDACs), including LMK235 and tubacin at clinically relevant concentrations, do not affect MK maturation. Furthermore, we show that HDACI-induced impairment of MK differentiation is associated with reduction of protein levels of the transcription factor GATA-1, but not tubulin hyperacetylation. Finally, we report that panHDACIs trigger a rapid loss of GATA-1 protein via a proteasome-dependent pathway. Our data support the notion that specifically targeting class II HDACs in cancer treatment is a potential strategy that would offer a safer alternative than current panHDACIs.

Overview of Glutamine Dependency and Metabolic Rescue Protocols.

Enhanced glutaminolysis and glycolysis are the two most remarkable biochemical features of cancer cell metabolism, reflecting increased utilization of glutamine and glucose in proliferating cells. Most solid tumors often outgrow the blood supply, resulting in a tumor microenvironment characterized by the depletion of glutamine, glucose, and oxygen. Whereas mechanisms by which cancer cells sense and metabolically adapt to hypoxia have been well characterized with a variety of cancer types, mechanisms by which different types of tumor cells respond to a dynamic change of glutamine availability and the underlying importance remains to be characterized. Here we describe the protocol, which uses cultured Hep3B cells as a model in determining glutamine-dependent proliferation, metabolite rescuing, and cellular responses to glutamine depletion. These protocols may be modified to study the metabolic roles of glutamine in other types of tumor or non-tumor cells as well.

Rapid AAV-Neutralizing Antibody Determination with a Cell-Binding Assay.

Recombinant adeno-associated virus (rAAV) has been developed as a successful vector for both basic research and human gene therapy. However, neutralizing antibodies (NAbs) against AAV capsids can abolish AAV infectivity on target cells, reducing the transduction efficacy. Absence of AAV NAb has become a prerequisite qualification for patients enrolled in gene therapy trials. Nevertheless, accurate assessment of AAV NAb has remained a challenging task. Here we developed a rapid assay based on the observations that AAV NAb inhibits rAAV binding to the host cell surface and NAb titers are negatively related to the amount of AAV genomes binding to the target cells. By quantifying the AAV genome on the target cells in the presence of anti-sera, AAV NAb titers can be accurately determined. The titer determined by this assay correlates well with the classical transduction-based assays. A major advantage of this method is that it can be carried out with a 30-min binding assay without the lengthy wait for a transduction outcome. This assay is independent of transduction performance of AAV serotype in the target cells. Therefore, the AAV cell-binding assay for NAb determination offers an alternative method for in vivo NAb assay.

Stanniocalcin 2 (STC2) expression promotes post-radiation survival, migration and invasion of nasopharyngeal carcinoma cells.

Background: Stanniocalcin 2 (STC2) expression is upregulated under multiple stress conditions including hypoxia, nutrient starvation and radiation. Overexpression of STC2 correlates with tumor progression and poor prognosis. Purpose: We previously demonstrated that overexpression of STC2 in nasopharyngeal carcinomas (NPC) positively correlates with radiation resistance and tumor metastasis, two major clinical obstacles to the improvement of NPC management. However, it remains elusive whether STC2 expression is a critical contributing factor for post-radiation survival and metastasis of NPC cells. Materials and methods: Using the radiation resistant CNE2 cell line as a model, we examined the importance of STC2 expression for post-radiation survival, migration and invasion. Here, we report the establishment of STC2 knockout lines (CNE2-STC2-KO) using the CRISPR/Cas9-based genome editing technique. Results: Compared with the parental line, STC2-KO cells showed similar proliferation and morphology in normal culture conditions, and loss of STC2 did not compromise the cell tumorigenicity in nude mice model. However, STC2-KO lines demonstrated increased sensitivity to X-radiation under either normoxic or hypoxic conditions. Particularly, upon X-radiation, parental CNE2 cells only slightly whereas STC2-KO cells remarkably decreased the migration and invasion ability. Cell cycle analysis revealed that loss of STC2 accumulated cells in G1 and G2/M phases but decreased S-population. Conclusion: These data indicate that the expression of STC2, which can be stimulated by metabolic or therapeutic stresses, is one important factor to promote survival and metastasis of post-radiation NPC cells. Therefore, targeting STC2 or relative downstream pathways may provide novel strategies to overcome radiation resistance and metastasis of NPC.

Carboxyl-terminal transactivation activity of hypoxia-inducible factor 1 alpha is governed by a von Hippel-Lindau protein-independent, hydroxylation-regulated association with p300/CBP.

Hypoxia-inducible factor 1 complex (HIF-1) plays a pivotal role in oxygen homeostasis and adaptation to hypoxia. Its function is controlled by both the protein stability and the transactivation activity of its alpha subunit, HIF-1 alpha. Hydroxylation of at least two prolyl residues in the oxygen-dependent degradation domain of HIF-1 alpha regulates its interaction with the von Hippel-Lindau protein (VHL) that targets HIF-1 alpha for ubiquitination and proteasomal degradation. Several prolyl hydroxylases have been found to specifically hydroxylate HIF-1 alpha. In this report, we investigated possible roles of VHL and hydroxylases in the regulation of the transactivation activity of the C-terminal activating domain (CAD) of HIF-1 alpha. We demonstrate that regulation of the transactivation activity of HIF-1 alpha CAD also involves hydroxylase activity but does not require functional VHL. In addition, stimulation of the CAD activity by a hydroxylase inhibitor, hypoxia, and desferrioxamine was severely blocked by the adenoviral oncoprotein E1A but not by an E1A mutant defective in targeting p300/CBP. We further demonstrate that a hydroxylase inhibitor, hypoxia, and desferrioxamine promote the functional and physical interaction between HIF-1 alpha CAD and p300/CBP in vivo. Taken together, our data provide evidence that hypoxia-regulated stabilization and transcriptional stimulation of HIF-1 alpha function are regulated through partially overlapping but distinguishable pathways.