Patent Application
20190022127
The present invention is related to the fields of cancer diagnosis, cancer therapy, prognosis predication of a cancer and determination of therapeutic regimen of a cancer. Particularly, the invention is related to LncHIFCAR (long noncoding HIF-1α co-activating RNA)/MIR31HG and its applications in cancer diagnosis, cancer therapy, prognosis predication of a cancer and determination of therapeutic regimen of a cancer.
Hypoxia is a common feature of rapidly growing solid tumors, tightly associated with tumor metastasis and poor prognosis, and a contributor to malignant progression and aggressive phenotype in many cancer types. Hypoxia-inducible factor-1 (HIF-1), a heterodimer consisting of α and β subunits, is a key regulator of the cellular response to hypoxia. Under hypoxic conditions, the HIF-1α subunit is stabilized and translocated to the nucleus where it forms a stable HIF-1 complex, specifically bound to the promoter regions of HIF-1 target genes and thereby inducing gene transcription. Proteins encoded by HIF-1 target genes are involved in multiple aspects of tumorigenesis, including glucose and energy metabolism, proliferation, cancer stem-like properties, angiogenesis, invasion and metastasis. The activation of HIF-1 pathways is associated with an aggressive tumor phenotype and poor clinical outcome in numerous cancer types, including oral cancer. For example, oral squamous cell carcinoma (OSCC) represents one of the most common malignancies worldwide with a high mortality rate mainly due to lack of early detection markers, frequent association with metastasis and aggressive phenotype.
Thus, there is a need to identify biomarkers and therapeutic targets for a cancer.
The present invention identifies a hypoxia-inducible lncRNA, LncHIFCAR (long noncoding HIF-1α co-activating RNA)/MIR31HG, and describes its oncogenic role as a HIF-1α co-activator that regulates the HIF-1 transcriptional network, crucial for cancer development. Extensive analyses of clinical data indicate LncHIFCAR level is substantially up-regulated in carcinoma (particularly, oral carcinoma), significantly associated with poor clinical outcomes and representing an independent prognostic predictor. Overexpression of LncHIFCAR induces pseudo-hypoxic gene signature, whereas knockdown of LncHIFCAR impairs the hypoxia-induced HIF-1α transactivation, sphere-forming ability, metabolic shift and metastatic potential in vitro and in vivo. Mechanistically, LncHIFCAR forms a complex with HIF-1α via direct binding, and facilitates the recruitment of HIF-1α and p300 cofactor to the target promoters. The invention uncovers a lncRNA-mediated mechanism for HIF-1 activation, and establishes the values of LncHIFCAR in diagnosis, prognosis and potential therapeutic strategy for carcinoma.
In one aspect, the invention provides a method of diagnosing whether a subject has, or is at risk for a cancer, a metastatic cancer or a primary cancer, comprising: (a) isolating a LncHIFCAR transcript in a biological sample from the subject; (b) measuring a test level of the isolated LncHIFCAR transcript; (c) comparing the test level to a control level of the LncHIFCAR transcript; and (d) determining a subject as having the cancer, metastatic cancer or primary cancer when the test level is higher than the control level.
In another aspect, the invention provides a method of determining a prognosis, recurrence-free survival or overall survival of a subject having, or suspected of a cancer, a metastatic cancer or a primary cancer, comprising: a) isolating a LncHIFCAR transcript in a biological sample from the subject; b) measuring a test level of the isolated LncHIFCAR transcript; c) comparing the test level to a control level of the LncHIFCAR transcript; and d) determining a subject as having a poor prognosis, poor recurrence-free survival or poor overall survival when the test level is higher than the control level.
In another aspect, the invention provides a kit for predicting a risk for developing an oral cancer, a metastatic oral cancer or a primary oral cancer or a prognosis, recurrence-free survival or overall survival of a subject, comprising reagents for determining a level of the LncHIFCAR in the sample.
In a further another aspect, the invention provides a method of treating an oral cancer, a metastatic oral cancer or a primary oral cancer in a subject comprising administering to the subject an effective amount of a therapeutic agent that blocks an expression or overexpression of MIR31HG gene or a physiological action of a LncHIFCAR transcript.
The invention found MIR3HG as a species that is significantly up-regulated in cancers. The invention describes a previously unrecognized role of the mature, spliced form of MIR31HG as a co-activator of HIF-1α that activates the pseudohypoxia signature required for hypoxia-induced metabolic reprogramming, sphere-forming ability and metastatic potential. As this RNA species does not contain miR-31 sequence and functions independently, it is defined as LncHIFCAR (long noncoding HIF-1α co-activating RNA). The invention also uncovers the up-regulation of LncHIFCAR/MIR31HG in oral carcinoma and the clinical relevance of LncHIFCAR as an independent adverse prognostic predictor for the cancer progression. Given its significance in the HIF-1 signaling pathway, LncHIFCAR/MIR31HG represents a novel and potential therapeutic target for the treatment of oral carcinoma.
The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. The singular forms “a,” “an,” and “the” refer to one or more than one, unless the context clearly dictates otherwise. For example, the term “comprising a nucleic acid molecule” includes single or plural nucleic acid molecules and is considered equivalent to the phrase “comprising at least one nucleic acid molecule.” The term “or” refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise. As used herein, “comprises” means “includes.” Thus, “comprising A or B,” means “including A, B, or A and B,” without excluding additional elements.
As used herein, the term “diagnosis” refers to a process of identifying a disease by its signs, symptoms and results of various tests. The conclusion reached through that process is also called “a diagnosis.” In some examples, a diagnosis includes determining whether a tumor is benign or malignant. In other examples, a diagnosis includes determining whether a subject with cancer has a good or poor prognosis.
As used herein, the term “prognosis” refers to the prediction of the likelihood of cancer-attributable death or progression, including recurrence, metastatic spread, and drug resistance, of a neoplastic disease. Poor prognosis can refer to any negative clinical outcome, such as, but not limited to, a decrease in likelihood of survival (such as overall survival, relapse-free survival, or metastasis-free survival), a decrease in the time of survival (e.g., less than 5 years, or less than one year), presence of a malignant tumor, an increase in the severity of disease, a decrease in response to therapy, an increase in tumor recurrence, an increase in metastasis, or the like. In particular examples, a poor prognosis is a decreased chance of survival.
As used herein, the term “independent Prognostic Factor” denotes the independent nature of a given prognostic factor is established by multivariate statistical analysis delineating its independence from other prognostic factors. Independent prognostic factors can be particularly useful in clinical medicine since, by their independence, they can be applied to various clinical scenarios and they can be relied upon even in the absence of other clinical information. As a consequence, the identification of independent prognostic factors is a major focus of oncologic study.
As used herein, the term “prediction” refers to the likelihood that a patient will respond either favorably or unfavorably to a drug or set of drugs, and also the extent of those responses, or that a patient will survive, following surgical removal or the primary tumor and/or chemotherapy for a certain period of time without cancer recurrence. The predictive methods of the present invention can be used clinically to make treatment decisions by choosing the appropriate treatment modalities for any particular patient. The predictive methods of the present invention are valuable tools in predicting if a patient is likely to respond favorably to a treatment regimen, such as surgical intervention, chemotherapy with a given drug or drug combination, and/or radiation therapy, or whether long-term survival of the patient, following surgery and/or termination of chemotherapy or other treatment modalities is likely.
As used herein, the term “expression” refers to the process by which the coded information of a gene is converted into an operational, non-operational, or structural part of a cell, such as the synthesis of a protein.
As used herein, the terms “overexpress”, “overexpression”, “overexpressed”, “up-regulate”, or “up-regulated” interchangeably refer to a biomarker that is transcribed or translated at a detectably greater level, usually in a cancer cell, in comparison to a non-cancer cell or cancer cell that is not associated with the worst or poorest prognosis. The term includes overexpression due to transcription, post transcriptional processing, translation, post-translational processing, cellular localization, and/or RNA and protein stability, as compared to a non-cancer cell or cancer cell that is not associated with the worst or poorest prognosis.
The terms “subject” and “individual” are defined herein to include animals, such as mammals, including, but not limited to, primates, cows, sheep, goats, horses, dogs, cats, rabbits, guinea pigs, rats, mice or other bovine, ovine, equine, canine, feline, rodent, or murine species. In a preferred embodiment, the animal is a human.
As used herein, the term “recurrence-free survival” includes (1) any recurrence (local or regional (including invasive ipsilateral tumor and invasive locol regional tumor), or distant) and (2) death due to any cause (both BC and non-BC causes of death).
As used herein, the terms “siRNA” and “short interfering RNA” are interchangeable and refer to single-stranded or double-stranded RNA molecules that are capable of inducing RNA interference. SiRNA molecules typically have a duplex region that is between 18 and 30 base pairs in length.
As used herein, the terms “piRNA” and “Piwi-interacting RNA” are interchangeable and refer to a class of small RNAs involved in gene silencing. PiRNA molecules typically are between 26 and 31 nucleotides in length.
As used herein, the terms “snRNA” and “small nuclear RNA” are interchangeable and refer to a class of small RNAs involved in a variety of processes including RNA splicing and regulation of transcription factors. The subclass of small nucleolar RNAs (snoRNAs) is also included.
As used herein, the term “biomarker” refers to a nucleic acid molecule which is present in a sample taken from interest subjects having human cancer as compared to a comparable sample taken from control subjects (e.g., a person with a negative diagnosis or undetectable cancer, normal or healthy subject).
As used herein, the term “risk” refers to the estimated chance of getting a disease during a certain time period.
As used herein, the term “treatment” is an intervention performed with the intention of preventing the development or altering the pathology or symptoms of a disorder.
As used herein, the term “level” refers to an absolute quantification of a molecule or an analyte in a sample, or to a relative quantification of a molecule or analyte in a sample, i.e., relative to another value such as relative to a reference or control taught herein, or to a range of values for the biomarker. These values or ranges can be obtained from a single subject or from a group of subjects.
In one aspect, the invention provides a method of diagnosing whether a subject has, or is at risk for a cancer, a metastatic cancer or a primary cancer, comprising:
In one embodiment, the method further includes a step of administering a siRNA silencing LncHIFCAR to treat the cancer. In some embodiments, the siRNA comprises a sequence selected from the group consisting of the following:
or its modified form.
The modified form of the siRNA includes, but is not limited to, 2′OMe nucleotides, 2′F nucleotides, 2′-deoxy nucleotides, 2′OMOE nucleotides, LNA nucleotides, and mixtures thereof. In preferred embodiments, the modified nucleotide comprises a 2′OMe nucleotide (e.g., 2′OMe purine and/or pyrimidine nucleotide) such as, for example, a 2′OMe-guanosine nucleotide, 2′OMe-uridine nucleotide, 2′OMe-adenosine nucleotide, 2′OMe-cytosine nucleotide, and mixtures thereof. In certain instances, the modified nucleotide is not a 2′OMe-cytosine nucleotide.
The siRNA can be prepared in many ways such as chemical synthesis, in vitro transcription, enzyme cleavage of long-chain dsRNA, vector expression of siRNA, PCR synthesis of siRNA expression elements. The presence of these methods provides a selection space for researchers and can be used to obtain better gene silencing efficiency.
The diagnosis described herein includes a diagnosis in various stages of a cancer. Examples of the stages include, but are not limited, early stage, invasion stage and metastatic stage of a cancer.
In another aspect, the invention provides a method of determining a prognosis, recurrence-free survival or overall survival of a subject having, or suspected of a cancer, a metastatic cancer or a primary cancer, comprising: a) isolating a LncHIFCAR transcript in a biological sample from the subject; b) measuring a test level of the isolated LncHIFCAR transcript; c) comparing the test level to a control level of the LncHIFCAR transcript; and d) determining a subject as having a poor prognosis, poor recurrence-free survival or poor overall survival when the test level is higher than the control level.
In one embodiment, the determination of a prognosis can be used as an independent prognostic factor.
In one embodiment, the method further includes a step of administering a siRNA silencing LncHIFCAR to treat the cancer. In some embodiments, the siRNA comprises a sequence of SEQ ID NO:1, 2, 3 or 4.
The level of LncHIFCAR transcript described herein can be measured in cells of a biological sample obtained from the subject. In some embodiments, the biological sample is a sample of tissue or fluid isolated from a subject, including but not limited to, for example, urine, blood, plasma, serum, fecal matter, bone marrow, bile, spinal fluid, lymph fluid, external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, organs, biopsies, and also samples containing cells or tissues derived from the subject and grown in culture, and in vitro cell culture constituents, including but not limited to, conditioned media resulting from the growth of cells and tissues in culture, recombinant cells, stem cells, and cell components. A corresponding control tissue or blood sample, or a control reference sample, can be obtained from unaffected tissues of the subject, from a normal human individual or population of normal individuals, or from cultured cells corresponding to the majority of cells in the subject's sample. The control tissue or blood sample is then processed along with the sample from the subject, so that the levels of LncHIFCAR from the subject's sample can be compared to the corresponding LncHIFCAR from the control sample. Alternatively, a control sample can be obtained and processed separately (e.g., at a different time) from the test sample and the level of LncHIFCAR produced from the sample can be compared to the corresponding LncHIFCAR level from the control sample.
The LncHIFCAR can be detected and quantitated by a variety of methods including, but not limited to, microarray analysis, polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), real time reverse transcriptase polymerase chain reaction (RT-PCR), quantitative real time polymerase chain reaction (qPCR), Northern blot, serial analysis of gene expression (SAGE), immunoassay, and mass spectrometry, any sequencing-based methods known in the art.
An increase in the level of LncHIFCAR in the sample obtained from the subject, relative to the level of a corresponding LncHIFCAR in a control sample, is indicative of the methods of the invention. The relative LncHIFCAR in the control samples can be determined with respect to one or more RNA expression standards. The standards can comprise, for example, the LncHIFCAR level in a standard cell line, the LncHIFCAR level in unaffected tissues of the subject.
In some embodiments, the test level and the control level may be expressed as a mean comparative quantification (Cq) test value and a mean comparative quantification (Cq) control value (delta Cq method). In such a case, the mean Cq test value and a mean Cq control value are normalized by an internal control.
In one embodiment, for determining the prognosis, recurrence-free survival or overall survival of a subject is, or is suspected of a cancer or diagnosing whether a subject has, or is at risk for developing a cancer, a metastatic cancer or a primary cancer, the level of the LncHIFCAR in the sample is greater than the reference level in the control sample.
In certain embodiments, the gene expressing LncHIFCAR used in the above methods is MIR31HG whose sequence is disclosed in NCBI Reference Sequence: NR_027054.1, NR_027054.2, NR_152877.1, NR_152878.1 or NR_152879.1.
In another further aspect, the invention provides a kit for predicting a risk for developing a cancer, a metastatic cancer or a primary cancer or a prognosis, recurrence-free survival or overall survival of a subject, comprising reagents for determining a level of the LncHIFCAR in the sample. The kit is assemblage of reagents for measuring RNA. It is typically in a package which contains all elements, optionally including instructions. The package may be divided so that components are not mixed until desired. The kit may contain reagents for determining RNA level.
In another aspect, the invention provides a method of treating a cancer, a metastatic cancer or a primary cancer in a subject comprising administering to the subject an effective amount of a therapeutic agent that blocks an expression or overexpression of MIR31HG gene or a physiological action of a LncHIFCAR transcript. In some embodiments, the therapeutic agent is selected from an antisense oligonucleotide, an antisense RNA, a small molecular inhibitor, an antisense cDNA, RNA, siRNA, esiRNA, shRNA, miRNA, decoy, RNA aptamer, RNA/DNA demethylating agent and RNA/DNA-binding protein/peptide or a compound to inhibit one or more physiological actions affected by LncHIFCAR. In one embodiment, the therapeutic agent is a siRNA silencing LncHIFCAR. In some embodiments, the siRNA comprises a sequence of SEQ ID NO: 1, 2, 3 or 4, or its modified form.
In some embodiments, the cancers described herein, including a tumor, metastasis, or other disease or disorder characterized by uncontrolled cell growth. In some embodiments, the cancers include, but are not limited to, oral cancer (such as an oral squamous cell carcinoma (OSCC)) or a hypoxia-mediated oral cancer), brain cancer (such as glioblastoma), kidney cancer (such as kidney renal clear cell carcinoma) or a hypoxia-mediated brain cancer, colorectal cancer or a hypoxia-mediated colorectal cancer, or uterine cancer (such as uterine corpus endometrial carcinoma) or a hypoxia-mediated uterine cancer. In other embodiments, the cancers described herein, carcinomas, myelomas, melanomas or gliomas may be treated.
All publications and patent documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent document were so denoted. By their citation of various references in this document, Applicants do not admit any particular reference is “prior art” to their invention.
Cell Culture
Cell lines HeLa and HEK293T (293T) were obtained from ATCC and cultured in DMEM supplemented with 10% (v/v) FBS (Gibco/Invitrogen). SAS cell line was obtained from JCRB cell bank and maintained in 1:1 mixture of DMEM and Ham's F12 medium (Gibco/Invitrogen) with 10% FBS. The oral cancer cell lines OC-2, OEC-M1 and HSC-3 were obtained and maintained as described previously. Cell lines were not authenticated, but regularly tested for mycoplasma contamination using Venor GeM Detection Kit (Minerva Biolabs). None of the cell line stock used in this study is found in the database of commonly misidentified cell lines listed by ICLAC. Transfection was performed using Lipofectamine 2000 (Invitrogen) or Fugene 6 (Roche Applied Science) as recommended by the manufacturer. To generate stable LncHIFCAR-knockdown SAS cell lines, SAS cells transduced with LncHIFCAR shRNA (sh-HIFCAR) or empty vector (sh-Ctrl) lentiviral particles were selected by Zeocin for 3 weeks. To generate luciferase-expressing and LncHIFCAR-knockdown SAS cell lines for xenograft study, the SAS stable clones (sh-Ctrl #1 and sh-HIFCAR #6) described above were transduced with a lentiviral vector containing firefly luciferase cDNA, which was constructed using the ViralPower Lentiviral Gateway Expression System from Invitrogen and selected by puromycin for 3 weeks. For hypoxia treatment, physical hypoxic conditions (1% oxygen) were generated by Forma™ Series II 3130 incubators (Thermo Scientific). Alternatively, to generate chemical-induced pseudo-hypoxic state, cells were treated with hypoxia-mimetic chemical cobalt chloride (Sigma-Aldrich) as indicated.
Real Time qRT-PCR
Total cellular RNA was isolated using Trizol (Invitrogen) reagent, and cDNA was generated using SuperScript II first-strand synthesis system (Invitrogen). Real-time quantitative PCR analysis was performed on the Bio-Rad iQ5 Real-Time PCR detection system (Bio-Rad) with Maxima SYBR Green qPCR Master Mix (Fermentas) or on the Rotor-Gene Q instrument (Qiagen) with QuantiFast SYBR Green PCR Kit (Qiagen). All expression levels, unless otherwise specified, were normalized against the GAPDH mRNA level. The primer sequences are listed in Supplementary Data 2.
Real time quantitative PCR for miR-31 was performed using miScript PCR Starter Kit and hsa-miR-31 miScript Primer Assay according to manufacturer's instructions (Qiagen).
Molecular Cloning and siRNA Transfection
To generate sense and anti-sense biotin-labelled LncHIFCAR transcripts, full-length LncHIFCAR cDNA was amplified from HeLa nuclear RNA and cloned into pCR2.1-TOPO (Invitrogen) by TA cloning. For in vitro transcription of biotin-labelled LncHIFCAR RNA deletion variants, the corresponding LncHIFCAR fragments were amplified and cloned into pGemT-Easy (Promega) by TA cloning. For ectopic overexpression of full-length RMRP, LncHIFCAR and LncHIFCAR (501-1500) in human cell lines, the PCR-generated DNA fragments containing the indicated regions were inserted into pCR2.1-TOPO by TA cloning, followed by sub-cloning into the EcoRI site of pSL-MS2 vector. To knockdown LncHIFCAR expression, DNA encoding shRNA specifically targeting LncHIFCAR at sequence GCTGCTGATGACGTAAAGT was cloned into pLenti4 vector. For siRNA-mediated knockdown of LncHIFCAR, two different siRNA oligonucleotides were synthesized and purified by Genepharma (Suzhou, Jiangsu, China). siRNAs were transfected at a final concentration of 20 nM using Lipofectamine RNAiMAX Reagent (Invitrogen) following the manufacturer's protocol. The sequences of siRNAs are listed in Supplementary Table 2.
The HIF-1α-expressing plasmid HA-HIF-1α-pcDNA3, as well as the reporter plasmid HRE-luciferase (HRE-FLuc) containing hypoxia-response elements (HREs) fused with a firefly luciferase, were purchased from Addgene. The pRL-SV40 luciferase constitutive reporter plasmid were purchased from Promega. To express and purify GST-HIF-1α, the PCR-amplified HIF-1α fragments from HA-HIF-1α-pcDNA3 was inserted into pGemT-Easy by TA cloning, followed by sub-cloning into the NotI site of pGEX-6p-1 plasmid (GE Healthcare). To purify GST-HIF-1α deletion variants, the corresponding amplified HIF-1α fragments were cloned into the BamHI/NotI sites of pGEX-6p-1. The integrity of each construct was verified by DNA sequencing and the sequences of specific primers designed for cloning. The pLK0.1-puro plasmid-based shRNAs, including TRCN0000003808 (HIF1A-sh1) and TRCN0000003809 (HIF1A-sh2), were obtained from the National RNAi Core Facility, Institute of Molecular Biology/Genomic Research Center, Academia Sinica, Taiwan.
cBioPortal and Oncomine Database Analysis
LncHIFCAR expression was analyzed using the Oncomine (www.oncomine.org) and cBioPortal (www.cbioportal.org) platforms. For the tumor versus normal analysis of LncHIFCAR (LOC554202) on Oncomine, the following datasets were used: Peng Head-Neck dataset (GEO: GSE25099)25, Vasko Thyroid dataset (GSE6004), He Thyroid dataset (GSE3467), Zhao Breast dataset (GSE3971) and TCGA colorectal dataset (TCGA Research Network; http://cancergenome.nih.gov/). The p value smaller than 0.05 was considered statistically significant. For LncHIFCAR/MIR31HG expression analysis and co-expression network discovery, the TCGA head and neck squamous cell carcinoma cohort (TCGA, Provisional) was analyzed on cBioPortal using the default options according to the instructions on the website. Pearson and Spearman correlations of the expression levels of 20532 genes in 522 HNSCC cases were accessed and computed by RNA-Seq V2 RSEM dataset. By default, only gene pairs with values >0.3 or <−0.3 in both measures are considered statistically significant and shown. Total 248 significantly co-expressed genes were listed, including LCAM and LDHA.
Cell Migration and Invasion Assay
3×104 cells were suspended in 100 μl of DMEM without FBS and seeded into the top chamber of 24-well plate-sized transwell inserts (BD Falcon, 353097) with a membrane of 8 μm pore size. The medium containing 10% FBS was placed into the lower chamber as a chemoattractant. After incubation for 24 hours, the cells that did not migrate through the pores were manually removed with a cotton swab. Cells presented at the bottom of the membrane were fixed and stained with crystal violet and then counted and imaged under microscope. Cell numbers were calculated in eight random fields for each chamber, and the average value was calculated. Each experiment was conducted in triplicate. Matrigel invasion assays were performed using Matrigel-coated transwell inserts with the procedure as described above.
Sphere Formation Assay
Single-cell suspensions of SAS cells were plated (1000 cells per well) into 6-well Ultra Low Attachment plates (Corning) in serum-free DMEM/F12 culture media (Gibco/Invitrogen) supplemented with 2% B27 (Invitrogen), 20 ng mL−1 bFGF (Invitrogen), and EGF (20 ng mL−1, Millipore). The cells were grown in a humidified atmosphere of 95% air and 5% CO2 for 15 days. Upon harvest, the spheres were counted (diameter >100 μm) with inverted phase contrast microscopy, followed by collection for RNA extraction. For pimonidazole staining, tumor spheres grown in normoxic suspension culture were allowed to attach to 0.1% gelatin-coated cover slips for 12 hours. Pimonidazole (Hypoxyprobe™-1 Kit, Hypoxyprobe, Burlington, USA) was applied to the spheres for 1 hour under normoxia. Intracellular pimonidazole complexes indicative of hypoxic conditions were detected by immunofluorescence microscopy using an anti-pimonidazole monoclonal antibody (1:200, Hypoxyprobe™-1 Kit). Cell nuclei were counterstained by Hoechst staining whereas necrotic cells were labeled with propidium iodide (PI) fluorescence staining. For quantification of necrotic spheres, suspended to tumor spheres were collected by centrifugation for the subsequent PI fluorescence staining.
Luciferase Reporter Assay
The HIF-1α-responsive luciferase construct (pHRE-FLuc) containing hypoxia-response elements (HREs) fused with a firefly luciferase was purchased from Addgen. For HRE luciferase assays, cells were seeded to 24-well plates at a density of 1×105 per well. After overnight incubation, cells were transiently co-transfected with the pHRE-FLuc reporter plasmid, empty vector, LncHIFCAR-expressing constructs or shRNA vector-targeting LncHIFCAR, as well as an internal control construct pRL-SV40 Renilla luciferase plasmid (Promega). 24 hours post transfection, the media was replaced and the cells were exposed to 20% or 1% 02 for 24 hours. At 48 hours post-transfection, cells were lysed with passive lysis buffer and assayed for firefly and Renilla luciferase activities using Varioskan Flash microplate luminometer (Thermo) with the Dual-Luciferase Assay System (Promega). All the luciferase activity were normalized against the Renilla values and expressed as the relative fold of control group.
Glucose Uptake and Lactate Production Assay
The intracellular glucose and extracellular lactate were measured with the fluorescence-based glucose assay and lactate assay kits (BioVision) according to the manufacturer's instructions, respectively. Vector control and LncHIFCAR-knockdown SAS clones were cultured for 24 hours following subsequent treatment of normoxia or hypoxia for 16 hours. Intracellular glucose levels and lactate levels in the culture media were measured and presented as folds relative to the level of control cells in normoxia. All measurement were normalized by cell number.
Nuclear and Cytoplasmic Fractionation
Subcellular fractionation of protein extracts from HeLa cells was performed using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) following the manufacturer's protocol. Nuclear/cytoplasmic fractionation of RNA was conducted with Nuclei EZ Lysis Buffer (Sigma), according to the manufacturer's protocol.
Biotinylated RNA Pull-Down
The biotinylated RNA pull-down assay was performed as described previously. Briefly, biotin-labeled RNAs was in vitro transcribed with AmpliScribe T7-Flash Biotin-RNA Transcription Kit (Epicentre), treated with RNase-free DNase I and purified with an RNeasy Mini Kit (Qiagen). The lambda transcript was generated with the control plasmid provided by the Transcription Kit. To form the proper secondary structure, biotinylated RNA supplied with RNA structure buffer (10 mM Tris pH 7, 0.1 M KCl and 10 mM MgCl2) was heated to 90° C. for 2 minutes, incubated on ice for 2 minutes, and then shifted to room temperature (RT) for 20 minutes. The RNA was then mixed with hypoxic HeLa nuclear extract or purified proteins and incubated at RT for one hour, followed by incubating with Streptavidin Mag Sepharose (GE Healthcare) at RT for one hour. After subsequent washes, the pull-down complexes were analyzed by standard western blot technique.
Antibodies and Western Blot Analysis
Cells were harvested, rinsed with PBS and lysed in lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1 mM EDTA, 1 mM MgCl2, 0.5% NP-40, 1 mM Na3VO4, 1 mM NaF, protease inhibitors cocktail). Cell lysates were separated on SDS-polyacrylamide gel, transferred to a PVDF membrane (Bio-Rad Laboratories) and immunoblotted using the following primary antibodies. Rabbit anti-HIF-1α (1:1000, GTX127309), anti-GST (1:5000, GTX110736), anti-lamin B2 (1:5000, GTX109894) and anti-tubulin (1:5000, GTX112141) antibodies, as well as mouse monoclonal anti-HIF-1α antibody (1:1000, GTX628480), were purchased from GeneTex. Mouse monoclonal antibodies recognizing β-actin (1:5000, A2228) were purchased from Sigma. Mouse monoclonal HIF-1β antibody [2B10] (1:2000, ab2771) were purchased from Abcam. Uncropped scans of the blots and gels are shown in Supplementary
Purification of GST-HIF-1α
E. coli host BL21(DE3) harboring the expression vector pGEX-6p-1-HIF-1α was cultured in Luria-Bertani medium with ampicillin (50 μg ml−1) and induced by 0.3 mM IPTG (isopropyl-β-D-thiogalactopyranoside) at 30° C. for 16 hours. Affinity purification of the recombinant protein was carried out with Pierce Glutathione Superflow Agarose (Pierce) following the manufacturer's instructions.
RNA Immunoprecipitation
RNA immunoprecipitation (RIP) was performed with the following modifications. Briefly, 2×107 of SAS cells treated with normoxia or hypoxia for 16 hours were crosslinked with 0.3% formaldehyde in medium for 10 min at 37° C., followed by neutralization with 125 mM glycine incubated at room temperature for 5 min. After two times wash with cold PBS, the cell pellets were lysed in RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Nonidet P-40, and 0.5% sodium deoxycholate, 0.5 mM DTT, RNase inhibitor and protease inhibitor cocktail), followed by sonication on ice and subsequent DNase treatment for 30 min. immunoprecipitation were performed by incubating protein A/G precleared nuclear lysates with a-HIF-1α antibody (1:100, NB100-105, Novus Biologicals) or equivalent mouse IgG (GTX35009, GeneTex) at 4° C. overnight. The RNA/antibody complex was then precipitated by incubation with protein A/G agarose beads. After subsequent wash following the standard protocol, the RNA samples were extracted with Trizol reagent (Invitrogen) and detected by qRT-PCR.
Immunoprecipitation
Vector control or LncHIFCAR knockdown SAS cells under hypoxic conditions for 24 hours were collected by centrifugation. The cell pellet was then resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 180 mM NaCl, 1% NP-40, protease and phosphatase inhibitor cocktail) and passed through a 21-gauge syringe several times. Immunoprecipitations were performed by incubating 1 mg protein A/G precleared cell lysates with 5 μg a-HIF-1α antibody (NB100-105, Novus Biologicals) or equivalent mouse IgG (GTX35009, GeneTex) at 4° C. for 3 hours. After subsequent wash, the immunoprecipated protein complex on the beads were analyzed by western blotting.
In Vitro RNA-Binding Assay
In vitro synthesized LncHIFCAR RNA supplied with RNA structure buffer (10 mM Tris pH 7, 0.1 M KCl and 10 mM MgCl2) was heated to 90° C. for 2 minutes, incubated on ice for 2 minutes, and then shifted to room temperature for 20 minutes to form the proper secondary structure. GST fusion proteins on glutathione-Sepharose beads (˜10 μl) were incubated with 2 μg in vitro synthesized LncHIFCAR RNA in 50 μl of RNA-binding buffer (50 mM Tris-HCl, pH 7.4, 100 mM KCl, 2 mM MgCl2, 0.1% NP-40. 1 mM DTT and ribonuclease inhibitor) for 30 min at 4° C. The beads were washed with RNA-binding buffer 3 times to remove unbound RNAs. The RNA samples retained on the beads were extracted with Trizol reagent (Invitrogen) and detected by qRT-PCR. The relative retention as to the input RNA level was calculated.
Chromatin Immunoprecipitation
Chromatin immunoprecipitation (ChIP) assays were performed mainly following the ChIP protocol from the University of California Davis Genome Center (http://farnham.genomecenter.ucdavis.edu). Briefly, vector control or LncHIFCAR knockdown SAS cell lines (2×107 cells per assay) treated with normoxia or hypoxia were cross-linked with 1% formaldehyde at 37° C. for 15 min and subsequently quenched in 125 mM glycine for 5 min. The cross-linked chromatin was sonicated to generate DNA fragments averaging 200-500 bp in length. Chromatin fragments were immunoprecipitated with antibodies against HIF-1α (1:100, NB100-105, Novus Biologicals), p300 (1:100, 05-257, Millipore), or equivalent mouse IgG (GTX35009, GeneTex). The precipitated DNA were purified using the QIAquick PCR Purification Kit (Qiagen) and analyzed by quantitative real-time PCR using the primers listed in Supplementary Data 2.
Chromatin Isolation by RNA Purification
Chromatin isolation by RNA purification (ChIRP) was performed using SAS cells adapting the protocols with minor modifications. Briefly, 20-mer antisense DNA probes targeting LncHIFCAR RNA, as well as the negative control lacZ RNA, were designed using the online probe designer at singlemoleculefish.com (http://www.singlemoleculefish.com/designer.html) as listed in Supplementary Table 4.
SAS cells treated with normoxia or hypoxia for 4 hours were crosslinked with 1% glutaldehyde for 10 min at room temperature with gentle shaking. Crosslinking was stopped with 125 mM glycine for 5 min. The cross-linked chromatin was incubated with the biotinylated DNA probes, subjected to streptavidin magnetic beads capturing and subsequent wash/elution steps essentially performed as described70. The eluted chromatin and RNA fragments were analyzed by qPCR using the primers listed in Supplementary Data 2.
Oral cancer SAS cell lines (sh-Ctrl#1 and sh-HIFCAR#6) used for metastasis model were all labeled with firefly luciferase as described above. In brief, 5×105 SAS cells (sh-Ctrl#1 and sh-HIFCAR#6, respectively) suspended in 0.5 ml of Matrigel (BD Bioscience) were injected into the tail veins of 6- to 8-week-old female athymic nude mice (nu/nu). Subsequently, the mice were monitored for metastases using the IVIS Lumina LT series III system (PerkinElmer) after intraperitoneal injection of luciferin once a week for 6 weeks after tail vein xenografting. Then, the lungs of the nude mice were excised post mortem for histology examination and hematoxylin-eosin staining. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. The animal studies were approved by National Health Research Institutes Institutional Animal Care and Use Committee (approval number: NHRI-IACUC-102078), and carried out under the institutional guidelines with animal welfare standards.
Patients and Clinical Samples
The research design, study protocols and information security were approved by the Institutional Review Board of Chi-Mei Medical Center (approval number: 10312-L07). Thereby, snap-frozen primary OSCC tissues and paired noncancerous mucosa tissues stored in liquid nitrogen were withdrawn from Chi-Mei Medical Center Tissue BioBank. Written informed consents were obtained from all participants.
Statistical Analysis
All data were expressed as mean±SD of three or more independent experiments. Sample sizes were selected based on experience from our previous publications. Samples were excluded only in the case where a technical error occurred during sample preparation or analysis. Statistical analyses were performed with SPSS software version 19.0 (Armonk). Differences between individual groups were analyzed by 2-tailed Student's t test. The survival curves were calculated using the Kaplan-Meier method, and the differences were assessed by a log-rank test. Statistical analyses of clinicopathological data were performed using Fisher's exact test. Univariate and multivariate Cox proportional hazards regression models were performed to identify the independent factors with a significant impact on patient survival. The hazard ratios (HRs) and 95% confidence intervals of the prognostic factors were calculated. The results were considered significant if P <0.05.
Data Availability
The TCGA and Oncomine data referenced during the study are available in a public repository from the cBioPortal for Cancer Genomics, TCGA (http://www.cbioportal.org/) and the Oncomine (www.oncomine.org) websites. For the tumor versus normal analysis of LncHIFCAR on Oncomine (search term: LOC554202), the following datasets were used: Peng Head-Neck dataset (Gene Expression Omnibus: GSE25099), Vasko Thyroid dataset (GSE6004), He Thyroid dataset (GSE3467), Zhao Breast dataset (GSE3971) and TCGA colorectal dataset (TCGA Research Network; http://cancergenome.nih.gov/). For LncHIFCAR expression analysis and co-expression network discovery, the TCGA head and neck squamous cell carcinoma cohort (TCGA, Provisional) was analyzed on cBioPortal platform (search term: MIR31HG). The authors declare that the data supporting the findings of this study are included within the article and its Supplementary Information files, or are available from the authors upon reasonable request.
To identify lncRNAs involved in HIF-1 signaling pathway and hypoxia-associated cancer progression, we initially selected 37 cancer-associated lncRNAs according to previous reports and examined their expression profiles in HeLa cells before and after hypoxia treatment using quantitative real-time PCR (qRT-PCR). Validation of a panel of known hypoxia-inducible protein-coding genes by qRT-PCR confirmed the robustness of our screenings. Compared with non-hypoxic controls, several lncRNAs with a >2-fold alteration in expression were identified under hypoxic conditions. (
To evaluate the clinical significance of LncHIFCAR in cancer progression, we first queried the Oncomine database (www.oncomine.com) to systematically assess the relative LncHIFCAR expression in different cancer types (normal versus cancer). Several types of cancer were found to exhibit a significant up-regulation of LncHIFCAR, including oral squamous cell carcinoma (OSCC, P=2.2×10−17, Student's t test;
To evaluate the possible role of LncHIFCAR in oral cancer, we first analyzed the level of LncHIFCAR in a panel of OSCC cell lines, OECM1, OC-2, HSC-3 and SAS. Compared to OEC-M1 and OC-2 cells established from primary OSCC tumors that exhibit minimal invasion capacity31, the highly invasive HSC-3 and SAS cells expressed higher LncHIFCAR level (
Cellular response to hypoxia is implicated in many critical aspects of cancer progression, including invasion, metastasis, stem properties maintenance and metabolism reprogramming. To further characterize the biological significance of LncHIFCAR in oral tumorigenesis, we knocked down this lncRNA with two independent small interfering RNAs (siRNAs). In addition, we used shRNA expressing-plasmids to generate stable LncHIFCAR knockdown clones (sh-HIFCAR) and vector controls (sh-Ctrl) in SAS cells, and examined the hypoxia-associated phenotypes in these cells. Notably, although miR-31 is located within the first intron of LncHIFCAR and shares the same transcription promoter, the LncHIFCAR knockdown shRNA/siRNA was designed to target the exon region, and thus only specifically downregulated LncHIFCAR but not miR-31, as determined by quantitative PCR (
HIF-1, consisting of HIF-1α and HIF-1β subunits, is the primary player driving cellular response to hypoxia by activating the expression of target genes involved in critical steps of phenotype changes in cancer progression, including angiogenesis (VEGF, vascular endothelial growth factor), mitochondrial function (BNIP3, BCL2/Adenovirus E1B 19 kDa interacting protein 3), metabolism reprogramming (GLUT1/SLC2A1, glucose transporter 1; LDHA,lactate dehydrogenase A; CA9, carbonic anhydrase 9; PDK1, pyruvate dehydrogenase kinase isozyme 1), invasion (LICAM, L1 cell adhesion molecule) and metastasis (LOXL2, lysyl oxidase homolog 2). Given its role in the hypoxia-associated cancer phenotypes, we next investigated the impact of LncHIFCAR on the expression of HIF-1 target genes. Strikingly, overexpression of LncHIFCAR but not RMRP, another hypoxia inducible lncRNA, induced the activation of HIF-1 target genes, known as pseudohypoxia, without a significant change of HIF1A mRNA in HeLa cells under normoxic condition (
Remarkably, transcriptional profiling of the tumor spheres suggested that the LncHIFCAR-mediated HIF-1 activation may functionally contribute to the sphere-forming ability in OSCC cells. When we examined the expression of LncHIFCAR and the HIF-1 target genes in HSC-3 and SAS parental and their sphere-forming cells, profound up-regulation of LncHIFCAR levels accompanied with HIF-1 target gene induction was detected in the sphere-forming cells (
To further validate the clinical relevance of LncHIFCAR with HIF-1 targets expression, we queried RNA sequencing and mRNA microarray data in published datasets from a variety of cancer studies. Congruent with our findings, HIF-1 target genes LDHA (Pearson's correlation r=0.41) and L1CAM (r=0.37) showed a positive correlation with the expression of LncHIFCAR in the TCGA HNSCC provisional cohort (n=522;
We next determined whether LncHIFCAR functions through a direct effect on HIF-1 transactivation potency. A HIF-1α reporter plasmid (HRE-FLuc) containing three HREs and firefly luciferase coding sequences was used for the promoter-activity assay. Ectopic expression of HIF-1α (3.62 fold) or LncHIFCAR (1.26 fold) alone increased HIF-1 transcriptional activity, while co-expression of HIF-1α and LncHIFCAR synergistically enhanced the promoter activity (6.94 fold;
The major mechanism of HIF-1 activation in hypoxia is attributed to the stabilization of HIF-1α protein. We found that neither overexpression nor knockdown of LncHIFCAR had a significant impact on the protein level of HIF-1α either under normoxic, chemical induced pseudo-hypoxic or physical hypoxic conditions (
Under normoxia conditions, the expression of HIF-1α proteins is generally low. We asked whether this low HIF-1α expression is required for LncHIFCAR-induced pseudohypoxia signature and HIF-1 transactivation as seen in
To delineate the structural determinants for the association between LncHIFCAR and HIF-1α, RNA pull-down assays were performed with a series of LncHIFCAR truncated fragments. Both the 5′-terminal (nucleotides 1-500) and 3′-terminal (nucleotides 1500-2166) regions were found to be associated with HIF-1α (
To verify whether LncHIFCAR directly acts on the target chromatins, we analyzed the loci of LncHIFCAR-dependent HIF-1 target genes as identified in
With our evidence of LncHIFCAR in HIF-1 activation and hypoxia-associated cancer phenotypes, the biological significance of LncHIFCAR in oral cancer progression in vivo was examined using mouse xenograft model. The vector control and LncHIFCAR knockdown SAS cells were genetically modified to express firefly luciferase, and subsequently injected intravenously into nude mice (Day 0). Following tail vein inoculation, the bioluminescence intensity of the lung region was measured weekly up to 6 weeks. While the vector control cells exhibited strong colonization to the lung with a timely progression, LncHIFCAR-knockdown cells showed a drastically reduced ability to colonize in lung (
| Number | Date | Country | |
|---|---|---|---|
| 62534901 | Jul 2017 | US |
