THE CIRCULAR RNA CIRCLTBP2 AS A BIOMARKER AND BIOTARGET IN INTRAHEPATIC CHOLANGIOCARCINOMAS FIELD OF THE INVENTION: The present invention is in the field of medicine, in particular oncology and hepatology. BACKGROUND OF THE INVENTION: Cholangiocarcinoma (CCA) includes a heterogeneous group of tumors that can occur anywhere in the biliary system. CCA is a rare cancer but both its incidence and mortality increased worldwide over the last decade. [1, 2] The silent presentation of CCA combined with its aggressive nature and resistance to chemotherapy, contributes to the high mortality rate of CCA, which represents 2% of all cancer-related deaths worldwide each year. The current methods for diagnosing CCA are not accurate enough, and the high heterogeneity of CCA severely limits the effectiveness of therapies. [3] Therefore, there is an urgent need of opening the field to new concepts to identify relevant therapeutic targets and biomarkers able to improve the survival of patients with CCA. [4, 5] Recently, circular RNAs (circRNAs) emerged as new regulators of cancer-related processes, such as cell proliferation, migration, and drug resistance, acting notably as sponges for microRNAs (miRNAs). [6] CircRNAs have been also found to be aberrantly expressed in several types of cancer, making them potential biomarkers for early detection and prognosis. These recent findings highlight the importance of circRNAs in cancer biology and their potential as targets for the development of new treatments. [7] However, there are currently very few studies in iCCA. [8, 9] For example, circACTN4 and circGGNBP2 were recently reported as regulator of iCCA progression modulating specific signaling pathways, including Hippo and Wnt/β-catenin. [10-12] Transforming Growth Factor beta (TGFβ) pathway also contributes to CCA carcinogenesis but the role of circRNAs as effectors of TGFβ remains unexplored so far in CCA. SUMMARY OF THE INVENTION: The present invention is defined by the claims. In particular, the present invention relates to the circular RNA circLTBP2 as a biomarker and biotarget in intrahepatic cholangiocarcinomas.
DETAILED DESCRIPTION OF THE INVENTION: Intrahepatic cholangiocarcinoma (iCCA) is a deadly cancer worldwide with an increasing incidence and limited therapeutic options. Therefore, there is an urgent need to open the field to new concepts for identifying clinically relevant therapeutic targets and biomarkers. Here, the inventors explored the role and the clinical relevance of circular RNA circLTBP2 in iCCA. In particular, CircLTBP2 (hsa-circ-0032603) was identified as a novel TGFβ-induced circRNA in several CCA cell lines. CircLTBP2 promotes tumor cell proliferation, migration and resistance to gemcitabine-induced apoptosis in vitro and tumor growth in vivo. Mechanistically, circLTBP2 acts as a competitive RNA regulating notably the activity of the tumor suppressor microRNA miR-338-3p, leading to the overexpression of its pro-metastatic targets. The restoration of miR-338-3p levels in iCCA cells reversed the pro-tumorigenic effects driven by circLTBP2, including the resistance to gemcitabine-induced apoptosis. In addition, circLTBP2 expression predicted a reduced survival, as detected in tumor tissues but also in serum exosomes isolated from patients with iCCA. In conclusion, CircLTBP2 is a novel effector of the pro- tumorigenic arm of TGFβ and a clinically relevant biomarker easily detected from liquid biopsies in iCCA. Methods of diagnosis: The first object of the present invention relates to a method of diagnosing an intrahepatic cholangiocarcinoma in a subject comprising determining the level of circular RNA circLTBP2 in a sample obtained from the patient wherein said level indicates whether the patient suffers or not from an intrahepatic cholangiocarcinoma. As used herein, the term “patient” or “subject” refers to any mammals, such as a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the patient is a human. In some embodiments, the patient is a human who is susceptible to have a intrahepatic cholangiocarcinoma as described above. As used herein, the term “cholangiocarcinoma” or “CCA” has its general meaning in the art and refers to a heterogeneous group of tumors that can occur anywhere in the biliary system. As used herein, the term “intrahepatic cholangiocarcinoma” or “iCCA” refers to a type of cholangiocarcinoma that occurs in the parts of the bile ducts within the liver.
As used herein term “diagnosing” refers to classifying a disease or a symptom, determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease and/or prospects of recovery. In the context of the invention, the method according to the invention allows to diagnose an intrahepatic cholangiocarcinoma. Typically, high levels of circLTBP2 indicate that the patient suffers from an intrahepatic cholangiocarcinoma, whereas low levels of circLTBP2 indicate that the patient does not suffer from an intrahepatic cholangiocarcinoma. As used herein, the term “high” refers to a measure that is greater than normal, greater than a standard such as a predetermined reference value or a subgroup measure or that is relatively greater than another subgroup measure. For example, high levels of circLTBP2 refers to a level of circLTBP2 that is greater than a normal circLTBP2 level. A normal circLTBP2 level may be determined according to any method available to one skilled in the art. High level of circLTBP2 may also refer to a level that is equal to or greater than a predetermined reference value, such as a predetermined cutoff. High level of circLTBP2 may also refer to a level of circLTBP2 wherein a high circLTBP2 subgroup has relatively greater levels of circLTBP2 than another subgroup. For example, without limitation, according to the present specification, two distinct patient subgroups can be created by dividing samples around a mathematically determined point, such as, without limitation, a median, thus creating a subgroup whose measure is high (i.e., higher than the median) and another subgroup whose measure is low. In some cases, a “high” level may comprise a range of level that is very high and a range of level that is “moderately high” where moderately high is a level that is greater than normal, but less than “very high”. As used herein, the term “low” refers to a level that is less than normal, less than a standard such as a predetermined reference value or a subgroup measure that is relatively less than another subgroup level. For example, low level of circLTBP2 means a level of circLTBP2 that is less than a normal level of in a particular set of samples of patients. A normal level of circLTBP2 measure may be determined according to any method available to one skilled in the art. Low level of circLTBP2 may also mean a level that is less than a predetermined reference value, such as a predetermined cutoff. Low level of circLTBP2 may also mean a level wherein a low level circLTBP2 subgroup is relatively lower than another subgroup. For example, without limitation, according to the present specification, two distinct patient subgroups can be
created by dividing samples around a mathematically determined point, such as, without limitation, a median, thus creating a group whose measure is low (i.e., less than the median) with respect to another group whose measure is high (i.e., greater than the median). As used herein, the term “predetermined reference value” refers to a threshold value or a cut- off value. A "threshold value", “reference value” or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement of the level of the marker of the invention (e.g. circLTBP2) in properly banked historical patient samples may be used in establishing the predetermined corresponding reference value. In some embodiments, the predetermined corresponding reference value is the median measured in the population of the patients for the marker of in the invention (circLTBP2 for example). In some embodiments, the threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after determining the level of the marker of the invention (circLTBP2 for example) in a group of reference, one can use algorithmic analysis for the statistic treatment of the levels determined in samples to be tested, and thus obtain a classification standard having significance for sample classification. The full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests. ROC curve is a comprehensive indicator the reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1-specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When
AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is quite high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER.SAS, CREATE-ROC.SAS, GB STAT VI0.0 (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc. Differential diagnosis: A further aspect of the invention relates to a method for discriminating an intrahepatic cholangiocarcinoma from a hepatocellular carcinoma in a subject comprising i) determining in a sample obtained from the patient the level of circLTBP2; ii) comparing the level determined at step i) with a predetermined reference value and iii) concluding that the patient has or is susceptible to have an intrahepatic cholangiocarcinoma when the level determined at step i) is higher than its predetermined reference value, or concluding that the patient has or is susceptible to have a hepatocellular carcinoma when the level determined at step i) is lower than its predetermined reference value. As used herein, the term “discriminating” refers to identify, observe a difference or distinguish two groups. Methods of predicting survival time: A further object of the present invention relates to a method of predicting the survival time of a patient suffering from an intrahepatic cholangiocarcinoma comprising determining the level of circular RNA circLTBP2 in a sample obtained from the patient wherein said level indicates the survival time. The method of the present invention is particularly suitable for predicting the duration of the overall survival (OS), progression-free survival (PFS), and/or the disease-free survival (DFS) of the cancer patient. Those of skill in the art will recognize that OS survival time is generally based on and expressed as the percentage of people who survive a certain type of cancer for a specific amount of time. Cancer statistics often use an overall five-year survival rate. In general, OS rates do not specify whether cancer survivors are still undergoing treatment at five years or
if they've become cancer-free (achieved remission). DFS (or relapse-free survival – RFS) gives more specific information and is the number of people with a particular cancer who achieve remission. Also, progression-free survival (PFS) rates (the number of people who still have cancer, but their disease does not progress) includes people who may have had some success with treatment, but the cancer has not disappeared completely. As used herein, the expression “short survival time” indicates that the patient will have a survival time that will be lower than the median (or mean) observed in the general population of patients suffering from said cancer. When the patient will have a short survival time, it is meant that the patient will have a “poor prognosis”. Inversely, the expression “long survival time” indicates that the patient will have a survival time that will be higher than the median (or mean) observed in the general population of patients suffering from said cancer. When the patient will have a long survival time, it is meant that the patient will have a “good prognosis”. Typically high levels of circLTBP2 correlate with short survival time whereas low levels of circLTBP2 correlate with long survival time. More particularly, the higher the level of circLTBP2 is, the shorter will be the survival time of the patient whereas, the lower the level of circLTBP2 is, the longer will be the survival time of the patient. In said embodiment, the predetermined reference value may be determined by carrying out a method comprising the steps of a) providing a collection of samples; b) providing, for each sample provided at step a), information relating to the actual clinical outcome for the corresponding patient (i.e. the duration of the survival); c) providing a serial of arbitrary quantification values; d) determining the level of circLTBP2 for each sample contained in the collection provided at step a); e) classifying said samples in two groups for one specific arbitrary quantification value provided at step c), respectively: (i) a first group comprising samples that exhibit a quantification value for level that is lower than the said arbitrary quantification value contained in the said serial of quantification values; (ii) a second group comprising samples that exhibit a quantification value for said level that is higher than the said arbitrary quantification value contained in the said serial of quantification values; whereby two groups of samples are obtained for the said specific quantification value, wherein the samples of each group are separately enumerated; f) calculating the statistical significance between (i) the quantification value obtained at step e) and (ii) the actual clinical outcome of the patients from which samples contained in the first and second groups defined at step f) derive; g)
reiterating steps f) and g) until every arbitrary quantification value provided at step d) is tested; h) setting the said predetermined reference value as consisting of the arbitrary quantification value for which the highest statistical significance (most significant) has been calculated at step g). For example the level of circLTBP2 has been assessed for 100 samples of 100 patients. The 100 samples are ranked according to the level of circLTBP2. Sample 1 has the highest level and sample 100 has the lowest level. A first grouping provides two subsets: on one side sample Nr 1 and on the other side the 99 other samples. The next grouping provides on one side samples 1 and 2 and on the other side the 98 remaining samples etc., until the last grouping: on one side samples 1 to 99 and on the other side sample Nr 100. According to the information relating to the actual clinical outcome for the corresponding patient, Kaplan Meier curves are prepared for each of the 99 groups of two subsets. Also for each of the 99 groups, the p value between both subsets was calculated. The predetermined reference value is then selected such as the discrimination based on the criterion of the minimum p value is the strongest. In other terms, the level of circLTBP2 corresponding to the boundary between both subsets for which the p value is minimum is considered as the predetermined reference value. Method of predicting the response to therapy: A further object of the present invention relates to a method of determining whether a patient suffering from an intrahepatic cholangiocarcinoma achieves a response to a therapy comprising determining the level of circular RNA circLTBP2 in a sample obtained from the patient wherein said level indicates whether the patient achieves or not a response to the therapy. Typically, low levels of circLTBP2 indicate that the patient achieves a response to the therapy whereas high levels of circLTBP2 indicate that the patient does not achieve a response to the therapy. In some embodiments, the predetermined level is the level determined in a sample obtained from the patient before the treatment. As used herein, the terms "achieve a response" or "respond" refer to the response to a therapy of the patient suffering from an intrahepatic cholangiocarcinoma. Typically such therapy induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to intrahepatic cholangiocarcinoma. In particular, in the context of the invention, the term "respond" refers to the ability of the therapy to an improvement of the pathological symptoms,
thus, the patient presents a clinical improvement compared to the patient who does not receive the therapy. The said patient is considered as a "responder" to the treatment. The term "not respond" refers to a patient who does not present any clinical improvement to the treatment with the therapy. This patient is considered as a "non-responder" to the therapy. Accordingly, the patient as considered "non-responder" has a particular monitoring in the therapeutic regimen. In some embodiments, the response to a treatment is determined by Response evaluation criteria in solid tumors (RECIST) criteria. This criteria refers to a set of published rules that define when the disease in the patients improve ("respond"), stay the same ("stabilize"), or worsen ("progress") during treatment. In the context of the invention, when the patient is identified as responder, it means that said patient improves overall and progression- free survival (OS/PFS). In particular, the method herein disclosed is particularly suitable for determining whether the achieves a response to chemotherapy. As used herein “chemotherapy” has its general meaning in the art and is a cancer treatment that uses drugs to stop the growth of cancer cells, either by killing the cells or by stopping them from dividing. The said drug can be for example a small molecule: small molecules which can be conveniently used for the invention include in particular genotoxic drugs. The standard chemotherapy drugs for cholangiocarcinoma are gemcitabine (Gemzar®) and cisplatin. Other drugs sometimes used include fluorouracil (also called 5-FU), oxaliplatin (Eloxatin®), and capecitabine (Xeloda®). Samples: As used herein, the term "sample" refers to any substance derived from a living organism. For example, a sample may be derived from blood as a urine sample, serum sample, a plasma sample, and or a whole blood sample. Alternatively, a sample may be derived from a tissue collected, for example, by a biopsy. In a particular embodiment, the sample has been previously obtained from the subject. Thus, in some embodiments, the methods of the present invention are performed in vitro or ex vivo.
In some embodiments, the biological is a tumor tissue sample. As used herein, the term “tumor tissue sample” means any tissue tumor sample derived from the patient. Said tissue sample is obtained for the purpose of the in vitro evaluation. In some embodiments, the tumor sample may result from the tumor resected from the patient. In some embodiments, the tumor sample may result from a biopsy performed in the primary tumour of the patient. The tumor tissue sample can be subjected to a variety of well-known post-collection preparative and storage techniques (e.g., fixation, storage, freezing, etc.) prior to determining the level of circLTBP2 of interest. Typically the tumor tissue sample is fixed in formalin and embedded in a rigid fixative, such as paraffin (wax) or epoxy, which is placed in a mould and later hardened to produce a block which is readily cut. The tumour tissue sample can be used in microarrays, called as tissue microarrays (TMAs). TMA consist of paraffin blocks in which up to 1000 separate tissue cores are assembled in array fashion to allow multiplex histological analysis. This technology allows rapid visualization of molecular targets in tissue specimens at a time, either at the DNA, RNA or protein level. TMA technology is described in WO2004000992, US8068988, Olli et al 2001 Human Molecular Genetics, Tzankov et al 2005, Elsevier; Kononen et al 1198; Nature Medicine. In some embodiments, the sample is a blood sample. As used herein the term “blood sample” means any blood sample derived from the subject. Collections of blood samples can be performed by methods well known to those skilled in the art. In some embodiments, the blood sample is a serum sample or a plasma sample. In some embodiments, the level of circLTBP2 is determined in the extracellular vesicles that are isolated from the blood sample of the patient. As used herein the term “extracellular vesicle” or “EV” has its general meaning in the art and denotes a plasma membrane vesicle shed from an apoptotic or activated cell. Circulating extracellular vesicles can be isolated from the blood sample by coupling filtration and optionally contacting them with a set of binding partners directed against the specific surface markers of said extracellular vesicles. Typically, the extracellular vesicles are isolated as described in the EXAMPLE.
Methods for measuring the level of circLTBP2: As used herein, the term “circular RNA” or “circRNA” refers to a non-coding RNA molecule harboring exons out of order from genomic context, a phenomenon termed “exon shuffling” or “non-colinear splicing”. CircRNA is thus a type of single-stranded RNA which, unlike linear RNA, forms a covalently closed continuous loop. In circular RNA, the 3' and 5' ends normally present in an RNA molecule have been joined together. Thus, a circRNA does not contain a free 3’-end or a free 5’ end, i.e. the entire nucleic acid is circularized. The circRNA is preferably a circularized, single stranded RNA molecule. Furthermore, the circRNA according to the present invention is a result of a head-to-tail splicing event that results in a discontinuous sequence with respect to the genomic sequence encoding the RNA. This means that a first sequence being present 5’-upstream of a second sequence in the genomic context, on the circRNA said first sequence at its 5’-end is linked to the 3’ end of said second sequence and thereby closing the circle. The consequence of this arrangement is that at the junction where the 5’-end of said first sequence is linked to the 3’-end of said second sequence a unique sequence is build that is neither present in the genomic context nor in the normally transcribed RNA, e.g. mRNA. These junctions in all identified circRNAs, in the genomic context, are flanked by the canonical splice sequence, the GT/AG splice signal known by the skilled person. The skilled person will recognize that a usual mRNA transcript contains exon-exon junctions in a tail-to-head arrangement, i.e. the 3’ end (tail) of exon being upstream in the genomic context is linked to the 5’ end (head) of the exon being downstream in the genomic context. The actual junction, i.e. the point at which the one exon is linked to the other is also referred to herein as “breakpoint”. This feature confers numerous properties to circular RNA, many of which have only recently been identified. For instance, recent studies have suggested that circRNAs play important regulatory roles in micro-RNA activity. As used herein, the term “circLTBP2” refers to the CircRNA available from the data base cirBase http://www.circbase.org/ under the accession number hsa_circ_0032603. In some embodiments, circLTBP2 is encoded by the nucleic acid sequences as set forth in SEQ ID NO:1. SEQ ID NO:1 > Homo sapiens latent transforming growth factor beta binding protein 2 (LTBP2), mRNA, NCBI Reference Sequence: NM_000428.3 1 ccaaaaataa aaccgtccgg gtccccttca gacggctgca ggcacaggga ggaggcgcga 61 aggtgcagca gccgtgcgag cccagctgga gtaggagcgc ggactcgagg ctcggggcgc 121 gcagccctcg ttccgccgag agccgggccc ccagtcggcc gcttcagggc cccctagact 181 cagagaagct ggccgccggg cggggccggg agaacagccc gcgggcgtcc agcgtgccga
241 ccacaaagct cttcgcggtg cccgcgcgca ccactctcca gccgccccgc gccatgaggc 301 cgcggaccaa agcccgcagc ccggggcgcg ccctgcggaa cccctggaga ggcttcctgc 361 cgctcaccct ggctctcttc gtgggcgcgg gtcatgccca aagggacccc gtagggagat 421 acgagccggc tggtggagac gcgaatcgac tgcggcgccc tgggggcagc tacccggcag 481 cggctgcagc caaggtgtac agtctgttcc gggagcagga cgcgcctgtc gcgggcttgc 541 agcccgtgga gcgggcccag ccgggctggg ggagccccag gaggcccacc gaggcggagg 601 ccaggaggcc gtcccgcgcg cagcagtcgc ggcgtgtcca gccacctgcg cagacccgga 661 gaagcactcc cctgggccag cagcaaccag caccccggac ccgggccgcg ccggctctcc 721 cacgcctggg gaccccacag cggtctgggg ctgcgccccc aaccccgccg cgagggcggc 781 tcacggggag gaacgtctgc gggggacagt gctgcccagg atggacaaca gcaaacagca 841 ccaaccactg tatcaaaccc gtttgcgagc cgccgtgcca gaaccggggc tcctgcagcc 901 gcccgcagct ctgtgtctgc cgctctggtt tccgtggagc ccgctgcgag gaggtcattc 961 ccgatgagga atttgacccc cagaactcca ggctggcacc tcgacgctgg gccgagcgtt 1021 cacccaacct gcgcaggagc agtgcggctg gagagggcac cttggccaga gcacagccgc 1081 cagcaccaca gtcgccgccc gcaccacagt cgccaccagc tgggaccctg agtggcctca 1141 gccagaccca cccttcccag cagcacgtgg ggttgtcccg cactgtccga cttcacccga 1201 ctgccacggc cagtagccag ctctcttcca acgccctgcc cccgggacca ggccttgagc 1261 agagagatgg cacccaacag gcggtacctc tggagcaccc ctcatccccc tgggggctga 1321 acctcacgga gaaaatcaag aagatcaaga tcgtcttcac tcccaccatc tgcaagcaga 1381 cctgtgcccg tggacactgt gccaacagct gtgagagggg cgacaccacc accctgtaca 1441 gccagggcgg ccatgggcac gatcccaagt ctggcttccg catctatttc tgccagatcc 1501 cctgcctgaa cggaggccgc tgcatcggca gggacgaatg ctggtgcccc gccaactcca 1561 ccgggaagtt ctgccacctg cctatcccgc agccggacag ggagcctcca gggagggggt 1621 cccgccccag ggccttgctg gaagccccac tgaagcagtc cactttcaca ctgccgctct 1681 ccaaccagct ggcctccgtg aacccctccc tggtgaaggt gcacattcac cacccacccg 1741 aggcctcagt gcagatccac caggtggccc aggtgcgggg cggggtggag gaggccctag 1801 tggagaacag cgtggagacc agacccccgc cctggctgcc tgccagccct ggccacagcc 1861 tctgggacag caacaacatc cctgctcggt ctggagagcc ccctcggcca ctgcccccag 1921 cagcacccag gcctcgagga ctgctgggcc ggtgttacct gaacactgtg aacggacagt 1981 gtgccaaccc tctgctggag ctgactaccc aggaggactg ctgtggcagt gtgggagcct 2041 tctggggggt gactttgtgt gccccatgcc cacccagacc agcctccccg gtgattgaga 2101 atggccagct ggagtgtcct caggggtaca agagactgaa cctcactcac tgccaagata 2161 tcaacgagtg cttgaccctg ggcctgtgca aggacgcgga gtgtgtgaat accaggggca 2221 gctacctgtg cacatgcaga cctggcctca tgctggatcc atcgcggagc cgctgtgtgt 2281 cggacaaggc aatctccatg ctgcagggac tgtgctaccg gtcgctgggg cccggcacct 2341 gcaccctgcc tttggcccag cggatcacca agcagatatg ctgctgcagc cgcgtgggca 2401 aagcatgggg cagcgagtgt gagaaatgcc ctctgcctgg cacagaggcc ttcagagaga 2461 tctgccctgc cggccacggc tacacctacg cgagctccga catccgcctg tccatgagga 2521 aagccgagga ggaggaactg gcaaggcccc caagggagca agggcagagg agcagcgggg 2581 cactgcccgg gccagcagag aggcagcccc tccgggtcgt cacggacacc tggcttgagg 2641 ccgggaccat ccctgacaag ggtgactctc aggctggcca ggtcacgacc agtgtcactc 2701 atgcacctgc ctgggtcaca gggaatgcca caaccccacc aatgcctgaa caggggattg 2761 cagagataca ggaagaacaa gtgaccccct ccactgatgt gctggtgacc ctgagcaccc 2821 caggcattga cagatgcgct gctggagcca ccaacgtctg tggccctgga acctgcgtga 2881 acctccccga tggatacaga tgtgtctgca gccctggcta ccagctgcac cccagccagg 2941 cctactgcac agatgacaac gagtgtctga gggacccctg caagggaaaa gggcgctgca 3001 tcaaccgcgt ggggtcctac tcctgcttct gctaccctgg ctacactctg gccacctcag 3061 gggcgacaca ggagtgtcaa gatatcaatg agtgtgagca gccaggggtg tgcagcgggg 3121 ggcagtgcac caacaccgag ggctcgtacc actgcgagtg tgatcagggc tacatcatgg 3181 tcaggaaagg acactgccaa gatatcaacg aatgccgtca ccccggtacc tgccctgatg 3241 ggagatgcgt caattcccct ggctcctaca cttgtctggc ctgtgaggag ggctaccggg 3301 gccagagtgg gagctgtgta gatgtgaatg agtgtctgac tcccggggtc tgtgcccatg 3361 gaaagtgcac caacctagaa ggctccttca gatgctcttg tgagcagggc tatgaggtca 3421 cctcagatga gaagggctgc caagatgtgg atgagtgtgc cagccgggcc tcatgcccca 3481 caggcctctg cctcaacacg gagggctcct tcgcctgctc tgcctgtgag aacgggtact 3541 gggtgaatga agacggcact gcctgtgaag acctagatga gtgtgccttc ccgggagtct 3601 gcccctccgg agtctgcacc aacacggctg gctccttctc ctgcaaggac tgcgatgggg 3661 gctaccggcc cagccccctg ggtgactcct gtgaagatgt ggatgaatgt gaagaccccc 3721 agagcagctg cctgggaggc gagtgcaaga acactgtggg ctcctaccag tgcctctgtc 3781 cccagggctt ccagctggcc aatggcaccg tgtgtgagga tgtgaatgag tgcatggggg 3841 aggagcactg cgcaccccac ggcgagtgcc tcaacagcca cgggtctttc ttctgtctgt
3901 gcgcgcctgg cttcgtcagc gcagaggggg gcaccagctg ccaggatgtg gacgagtgtg 3961 ccaccacaga cccgtgtgtg ggagggcact gtgtcaacac cgagggctcc ttcaactgtc 4021 tatgtgagac tggcttccag ccctccccag agagtggaga gtgtgtggat attgacgagt 4081 gtgaggacta tggagacccg gtgtgtggca cctggaagtg tgaaaacagc cctggctcct 4141 accgctgtgt tctgggctgc cagcctggct tccacatggc cccgaacgga gactgcattg 4201 acatagacga gtgcgccaac gacaccatgt gtggcagcca cggcttctgt gacaacactg 4261 atggctcctt ccgctgcctc tgtgaccagg gcttcgagat ctctccctca ggctgggact 4321 gtgtggatgt gaacgagtgt gagcttatgc tggcggtatg tggggccgcg ctctgtgaga 4381 acgtggaggg ctccttcctg tgcctctgtg ccagtgacct ggaggagtac gatgcccagg 4441 aggggcactg ccgcccacgg ggggctggag gtcagagtat gtctgaggcc ccaacggggg 4501 accatgcccc ggcccccacc cgcatggact gctactccgg gcagaagggc catgcgccct 4561 gctccagtgt cctgggccgg aacaccacac aggctgaatg ctgctgcacc cagggcgcta 4621 gctggggaga tgcctgtgac ctctgcccgt ctgaggactc agctgaattc agcgagatct 4681 gccctagtgg aaaaggctac attcctgtgg aaggagcctg gacgtttgga cagaccatgt 4741 acacagatgc ggatgagtgt gtgatattcg ggcctggtct ctgcccgaac ggccggtgcc 4801 tcaacaccgt gcctggttat gtctgcctgt gcaatcccgg cttccactac gatgcttccc 4861 acaagaagtg tgaggatcac gatgagtgcc aggacctggc ctgtgagaat ggcgagtgcg 4921 tcaacacgga gggctccttc cactgcttct gcagcccccc gctcaccctg gacctcagcc 4981 agcagcgctg catgaacagc accagcagca cggaggacct ccctgaccac gacatccaca 5041 tggacatctg ctggaaaaaa gtcaccaatg atgtgtgcag cgaacccctg cgtgggcacc 5101 gcaccaccta cacggaatgc tgctgccagg acggcgaggc ctggagccag cagtgtgctc 5161 tgtgtccccc gaggagctct gaggtctatg ctcagctgtg caacgtggct cgcattgagg 5221 cagagcggga ggccggggtc cacttccggc caggctatga gtatggcccc gggcccgatg 5281 acctgcacta cagcatctat ggcccagatg gggccccctt ctacaactac ctgggccccg 5341 aggacaccgt ccctgagcct gccttcccca acacagccgg tcactcagcg gaccgcacac 5401 ccatccttga gtctcctttg cagccctcag aactccagcc ccactacgtg gccagccatc 5461 cagagccccc agccggcttc gaagggcttc aggcggagga gtgcggcatc ctgaacggct 5521 gtgagaatgg ccgctgtgtg cgcgtgcggg agggctacac ctgtgactgt tttgagggct 5581 tccagctgga tgcggcccac atggcctgcg tagatgtgaa tgagtgtgat gacttgaacg 5641 ggcctgctgt gctctgtgtc catggttact gcgagaacac agagggctcc taccgctgcc 5701 actgctcccc gggatatgtg gctgaggcag ggccccccca ctgcactgcc aaggagtagc 5761 agtcaggggt cagtgtggca actacctgga aatggcctcc agtcacaggc aggggccttg 5821 aggatgattt cctagctggg aagacaccgt gacatcaggc cagaggtttc caatcagcct 5881 tgcctgcttt catctctccc agcttagcct ctggctgtaa gcttcggtca ttgcctccat 5941 gcccttgctt ggctcaagca ccaccaatcg ctttaatgct tcagccaccg catgaggccc 6001 tgtccaccac ctttcctggc cttgctatgg gatgcttacc aaaggatggc cctcatccac 6061 cctcccaagc tgtgcgagca tgcaaggccc catggcctca cactgcagac acccctttcc 6121 agccacaatc caccatcatc ctgacgatcc cacaactggg acagaggcta catctgccct 6181 agggaggtcc ttcagaatct gtggagcaag aaaggatttg gggaagcttg gggactgact 6241 ccagagcccc ctcctaagaa ccatcaccac cactcagcca atctgttctg ggccctgatt 6301 ttgccacacc tccatcctgt agcccattct ctgaccccaa ggagtggcag aagatccctt 6361 cactcagaga agcaaggctg atattagctt gttgaatgta agagacacaa atgaagaaga 6421 acaaagagcc tgagaaagca gcaagaggac atgatgaaaa atacgtggag ttgatgagaa 6481 aggggagcca aggctttata cgtctaaaga aaatattcag tagctgaatc cgcccagtga 6541 tagcctgtgg gcaccagcag caagggctgc catgggatac agcacccatc tacaaagacc 6601 tctattacat aaacactgct tcttacagga aacaaacctc ttctgggatc tccttttgtg 6661 aaaaccagtt tgatgtgcta aaagtaaaaa gtctattttc cagtgtggtc ttgttcagaa 6721 gcagccagat ttccaatgtt gtttttcccc tccactcaga aacccctgcc ctttcccttc 6781 agaaaacgat ggcaggcatt cctctgagtt tacaagcaga gactcactcc aacccaaact 6841 agctgggagt tcagaaccat ggtggaataa agaaatgtgc atctggtctc ttctgttgtt 6901 tttatttcat atcagattaa atttctttac catgttggct aagtctaaat attagagatg 6961 aggctgtgcc tactccctgg ccagctctgc tgatagccta tgatgggttc caatgggaaa 7021 tgactcttta ctattaaaag acaaggaaag ctctgacttc gtacttctct gatgaatggc 7081 aatgtaaatg aacaaggctc catgtgactg gagcatggaa gtgaatgcta ctttcttaat 7141 ttaatctgcc ctgtcctacc tgctcctctg attgttagcc atcacataac ttattgaatg 7201 cttgccatgt gccaggcact gtgctgagtg ccatacatac atttcattta attatccaat 7261 aatcctactt actattgttt attctcaatt tacaggtgag gaaactgaga catagaaagc 7321 ttaaataatt tgctctaggt ccctatacta attgagatgt tttcagcaga caaaaataag 7381 tcactcttac gcatgtaaaa atattctttc actcataata aaagaaatgc aacttaaaac 7441 catactgaga tcctttttaa aactgtcaga ttggcaaaga tcaaaaagct tgttaagact 7501 cctagctggc aagcatgtgg gaaataggta ctctcataac agctgacaga agtacatatt
7561 gaagaggaca atatggcaat gtccttcaaa attataaaag cactaactct ttgatccagt 7621 agtttccact tctaggaact taaccttcag atatattttc accagatata aaatgacata 7681 cgtaagaagt tattactgca tcatttgtaa tagctaaaga ttagaaacaa ttaattgcta 7741 tggaaccctt aaaaaaggtg gctctaaatt ccttaagaaa gatatccaaa acaaattaag 7801 gggaaaaggg agggttgcat aacagcatgt aacataacac tatttgtcta aagaatgggg 7861 aaagagaagc ggttgtctgt tcacctcatc cttgccactc cgccttatac ctcaccatcg 7921 gagaacttac catcccccaa atatatcctg ctggcactac accttcgtaa ctgctgttcc 7981 cactgccccc acaacactcc aatctccacc ttcctgccaa ccttgagata ttgtatgcat 8041 atatacatat ttcctttgtt taaaatacaa atagcatact atgcatgctg ttctgcactt 8101 tgcttttttt ttatacttgc aattagagct gcctcatttc attttacagc tgtatactat 8161 tccattgaat ggataaacca taatttattt aatcagtccc tcactgacag acgtttccat 8221 tgtttcctgt cttttgctat aaataatgct gtaatgaata ttcttgtgca tatttcacat 8281 taatttatgg gattacatct gcaggataaa ttcttagaac caatggtggt ttgtggggag 8341 ggtgacaggg tggataggta caggattggg agacttttca ctgtatctcc ttttgtattt 8401 ttttgaattt tgtaccatat gtatcaccta ttcaaaaaaa ataaaatatt tttaaataca According to the invention, measuring the level of circLTBP2 in the sample obtained from the patient can be performed by a variety of techniques. For example the nucleic acid contained in the samples is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid- binding resins following the manufacturer's instructions. Conventional methods and reagents for isolating RNA from a sample comprise High Pure miRNA Isolation Kit (Roche), Trizol (Invitrogen), Guanidinium thiocyanate-phenol-chloroform extraction, PureLink™ miRNA isolation kit (Invitrogen), PureLink Micro-to- Midi Total RNA Purification System (invitrogen), RNeasy kit (Qiagen), miRNeasy kit (Qiagen), Oligotex kit (Qiagen), phenol extraction, phenol-chloroform extraction, TCA/acetone precipitation, ethanol precipitation, Column purification, Silica gel membrane purification, PureYield™ RNA Midiprep (Promega), PolyATtract System 1000 (Promega), Maxwell® 16 System (Promega), SV Total RNA Isolation (Promega), geneMAG-RNA / DNA kit (Chemicell), TRI Reagent® (Ambion), RNAqueous Kit (Ambion), ToTALLY RNA™ Kit (Ambion), Poly(A)Purist™ Kit (Ambion) and any other methods, commercially available or not, known to the skilled person. Although circRNA detection in sample is possible without any preprocessing of the total RNA sample, it is preferred to deplete ribosomal RNAs (rRNA), preferably the majority of rRNA, to increase the sensitivity of circRNA detection, in particular when using RNA Sequencing approaches. To this end, the content of rRNA in the sample should be depleted to less than 20%, preferably less than 10%, more preferably less than 2% with respect to the total RNA content. The rRNA depletion may performed as known in the art, e.g. it may be facilitated by commercially available kits (e.g. Ribominus, Themo Scientific) or enzymatic methods (Xian Adiconis et al.
Comprehensive comparative analysis of RNA sequencing methods for degraded or low input samples Nat Methods.2013 July; 10(7): 10.1038/nmeth.2483.). The level of circLTBP2 in the sample may be determined by any suitable method. Any reliable method for measuring the level or amount of circRNA in a sample may be used. Generally, circRNA can be detected and quantified from a sample (including fractions thereof), such as samples of isolated RNA by various methods known for mRNA, including, for example, amplification-based methods (e.g., Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), rolling circle amplification, etc.), hybridization-based methods (e.g., hybridization arrays (e.g., microarrays), NanoString analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, in situ hybridization, etc.), and sequencing-based methods (e.g., next- generation sequencing methods, for example, using the Illumina or IonTorrent platforms). Other exemplary techniques include ribonuclease protection assay (RPA) and mass spectroscopy. The circRNA according to the present invention may be detected using different techniques. As outlined herein, the exon-exon junction in a head-to-tail arrangement is unique to the circRNAs. Hence, the detection of these is preferred. Nucleic acid detection methods are commonly known to the skilled person and include probe hybridization based methods, nucleic acid amplification based methods, and nucleic acid sequencing, or combinations thereof. Hence, in a some embodiments circRNA is detected using a method selected from the group consisting of probe hybridization based methods, nucleic acid amplification based methods, and nucleic acid sequencing. Probe hybridization based method employ the feature of nucleic acids to specifically hybridize to a complementary strand. To this end nucleic acid probes may be employed that specifically hybridize to the exon-exon junction in a head-to-tail arrangement of the circRNA, i.e. to a sequence spanning the exon-exon junction, preferably to the region extending from 10 nt upstream to 10 nt downstream of the exon-exon junction, preferably to the region from 20 nt upstream to 20 nt downstream of the exon-exon junction, or even a greater region spanning the exon-exon junction. The skilled person will recognize that hybridization probes specifically hybridizing to the respective sequence of the circRNA may be used, as well as hybridization probes specifically hybridizing to the reverse complement sequence thereof, e.g. in case the circRNA is previously reverse transcribed to cDNA and/or amplified.
Hybridization can also be used as a measure of homology between two nucleic acid sequences. A nucleic acid sequence hybridizing specifically to an exon-exon junction in a head-to-tail arrangement according to the present invention may be used as a hybridization probe according to standard hybridization techniques. The hybridization of the probe to DNA or RNA from a test source (e.g., the bodily fluid, like whole blood, or amplified nucleic acids from the sample of the bodily fluid) is an indication of the presence of the relevant circRNA in the test source. Hybridization conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 6.3.1-6.3.6, 1991. Preferably, specific hybridization refers to hybridization under stringent conditions. “Stringent conditions” are defined as equivalent to hybridization in 6× sodium chloride/sodium citrate (SSC) at 45° C., followed by a wash in 0.2×SSC, 0.1% SDS at 65° C.; or as equivalent to hybridization in commercially available hybridization buffers (e.g. ULTRAHyb, ThermoScientific) for blotting techniques and 5×SSC 0.5% SDS (750 mM NaCl, 75 mM sodium citrate, 0.5% sodiumdodecylsulfate, pH 7.0) for array based detection methods at 65° C. The means and methods of the present invention preferably comprise the use of nucleic acid probes. A nucleic acid “probe” according to the present invention is an oligonucleotide, nucleic acid or a fragment thereof, which is substantially complementary to a specific nucleic acid sequence. “substantially complementary” refers to the ability to hybridize to the specific nucleic acid sequence under stringent conditions. The skilled person knows means and methods to determine the levels of nucleic acids in a sample and compare them to control levels. Such methods may employ labeled nucleic acid probes according to the invention. “Labels” include fluorescent or enzymatic active labels as further defined herein below. Such methods include real-time PCR methods and microarray methods, like Affimetrix®, nanostring and the like. The determination of the circRNAs or their level may also be detected using sequencing techniques. The skilled person is able to use sequencing techniques in connection with the present invention. Sequencing techniques include but are not limited to Maxam-Gilbert Sequencing, Sanger sequencing (chain-termination method using ddNTPs), and next generation sequencing methods, like massively parallel signature sequencing (MPSS), polony sequencing, 454 pyrosequencing, Illumina (Solexa) sequencing, SOLiD sequencing, or ion torrent semiconductor sequencing or single molecule, real-time technology sequencing (SMRT). Typically, the level of a circRNA is determined by detection of an exon-exon-junction in a
head-to-tail arrangement. The detection of circular RNA has been previously described in Memczak S, Jens M, Elefsinioti A, et al. Circular RNAs are a large class of animal RNAs with regulatory potency. Nature. 2013; 495(7441):333-338, which is incorporated herein by reference in particular as relates to the detection and annotation of circRNAs. The biogenesis of many mammalian circRNAs depends on complementary sequences within flanking introns Hence, in some embodiments, the two introns upstream and downstream of and direct adjacent in the genomic context to the exons of the exon-exon junction (i.e. forming the exon-exon junction) in a head to tail arrangement often contain complementary sequences, e.g. a complementary sequence stretch of at least 15 nucleotides, preferably 500 nucleotides, more preferably 1000 nucleotides. For detection of circRNA in principle, the RNA of a sample is sequenced after reverse transcription and library preparation. Afterwards, the sequences are analyzed for the presence of exon-exon junctions in a head-to-tail arrangement. Further, in some embodiments, RNA sequences which map continuously to the genome by aligning without any trimming (end-to-end mode) are neglected. Reads not mapping continuously to the genome are preferably used for circRNA candidate detection. The terminal sequences (anchors) from the sequences, e.g.20 nt or more, may be extracted and re-aligned independently to the genome. From this alignment the sequences may be extended until the full circRNA sequence is covered, i.e. aligned. Consecutively aligning anchors indicate linear splicing events whereas alignment in reverse orientation indicates head-to-tail splicing as observed in circRNAs. The so identified resulting splicing events are filtered using the following criteria 1) GT/AG signal flanking the splice sites in the genomic context; 2) the breakpoint, i.e. the exon-exon-junction can be unambiguously detected; and 3) no more than 100 kilobases distance between the two splice sites in the genomic context. Furthermore, further optional criteria may be used, depending on the method chosen; e.g. a maximum of two mismatches when extending the anchor alignments; a breakpoint no more than two nucleotides inside the alignment of the anchors; at least two independent reads supporting the head-to-tail splice junction; and/or a minimum difference of 35 in the bowtie2 alignment score between the first and the second best alignment of each anchor. The detection/determination of the circRNAs and the respective level may also employ nucleic acid amplification method alone or in combination with the sequencing and/or hybridization method. Nucleic acid amplification may be used to amplify the sequence of interest prior to detection. It may however also be used for quantifying a nucleic acid, e.g. by real-time PCR methods. Such methods are commonly known to the skilled person. Nucleic acid amplification
methods for example include rolling circle amplification (such as in Liu, et al., “Rolling circle DNA synthesis: Small circular oligonucleotides as efficient templates for DNA polymerases,” J. Am. Chem. Soc.118:1587-1594 (1996).), isothermal amplification (such as in Walker, et al., “Strand displacement amplification—an isothermal, in vitro DNA amplification technique,” Nucleic Acids Res. 20(7):1691-6 (1992)), ligase chain reaction (such as in Landegren, et al., “A Ligase-Mediated Gene Detection Technique,” Science 241:1077-1080, 1988, or, in Wiedmann, et al., “Ligase Chain Reaction (LCR)—Overview and Applications,” PCR Methods and Applications (Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, N Y, 1994) pp. S51-S64.)). Nucleic-acid amplification can be accomplished by any of the various nucleic-acid amplification methods known in the art, including but not limited to the polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification system (TAS), nucleic acid sequence based amplification (NASBA), rolling circle amplification (RCA), transcription-mediated amplification (TMA), self-sustaining sequence replication (3SR) and Qβ amplification. It will be readily understood that the amplification of the circRNA may start with a reverse transcription of the RNA into complementary DNA (cDNA), optionally followed by amplification of the so produced cDNA. Measuring the level of miR-338-3p: In some embodiments, the methods of the present invention further comprises determining the level of miR-338-3p in the sample obtained from the subject wherein the combined measures of circLTBP2 and miR-338-3p indicate whether: - the subject suffers or not from an intrahepatic cholangiocarcinoma, or - the subject will have a long or short survival time, or - the subject achieves or not a response to the therapy. Typically, high levels of circLTBP2 and low levels of miR-338-3p indicate that: - the subject suffers from an intrahepatic cholangiocarcinoma, or - the subject will have a short survival time - the subject does not achieve a response to the therapy. Typically, low levels of circLTBP2 and high levels of miR-338-3p indicate that: - the subject does not suffer from an intrahepatic cholangiocarcinoma, or - the subject will have a long survival time
- the subject achieves a response to the therapy. As used herein, the term “microRNA” or “miRNA” refers to an RNA molecule that is approximately 21-23 nucleotides (nt) in length. miRNAs can range between 18-26 nucleotides in length. Typically, miRNAs are single-stranded. However, in some embodiments, miRNAs may be at least partially double-stranded. In some embodiments, miRNAs may comprise an RNA duplex (referred to herein as a “duplex region”) and may optionally further comprises one or two single-stranded overhangs. In some embodiments, the miRNA comprises a duplex region ranging from 15 to 29 by in length and optionally further comprising one or two single- stranded overhangs. A miRNA may be formed from two RNA molecules that hybridize together or may alternatively be generated from a single RNA molecule that includes a self-hybridizing portion. In general, free 5’ends of miRNA molecules have phosphate groups, and free 3’ ends have hydroxyl groups. The duplex portion of a miRNA usually, but does not necessarily, comprise one or more bulges consisting of one or more unpaired nucleotides. One strand of a miRNA includes a portion that hybridizes with a target RNA. In some embodiments, one strand of the miRNA is not precisely complementary with a region of the target RNA, meaning that the miRNA hybridizes to the target RNA with one or more mismatches. In some embodiments, one strand of the miRNA is precisely complementary with a region of the target RNA, meaning that the miRNA hybridizes to the target RNA with no mismatches. Typically, miRNAs are thought to mediate inhibition of gene expression by inhibiting translation of target transcripts. However, in some embodiments, miRNAs may mediate inhibition of gene expression by causing degradation of target transcripts. As used herein, the term “miR-338-3p” has its general meaning in the art and refers to the miRNA available from the data base http://mirbase.org under the miRBase accession number MIMAT0000763 (hsa-miR-338-3p). The nucleic acid sequence of miR-338-3p is represented by SEQ ID NO:2. SEQ ID NO:2 >hsa-miR-338-3p MIMAT0000763 UCCAGCAUCAGUGAUUUUGUUG The expression level of miR-338-3p in the sample may be determined by any suitable method. Any reliable method for measuring the level or amount of miRNA in a sample may be used. Generally, miRNA can be detected and quantified from a sample (including fractions thereof), such as samples of isolated RNA by various methods known for mRNA, including, for example,
amplification-based methods (e.g., Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), rolling circle amplification, etc.), hybridization-based methods (e.g., hybridization arrays (e.g., microarrays), NanoString analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, in situ hybridization, etc.), and sequencing-based methods (e.g., next- generation sequencing methods, for example, using the Illumina or IonTorrent platforms). Other exemplary techniques include ribonuclease protection assay (RPA) and mass spectroscopy. Methods of therapy: Further aspect of the present invention relates to methods of treating intrahepatic cholangiocarcinoma. Typically, the methods of therapy as herein disclosed a carried out once the patient has been diagnosed according to the methods of diagnosis according to the present invention. As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period"
refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]). circRNA inhibitors: In particular, a further object of the present invention relates to a method of treating an intrahepatic cholangiocarcinoma in a patient in need thereof comprising administering to the patient an circRNA inhibitor of circLTBP2. As used herein, the term “circRNA inhibitor” refers to any compound that blocks circRNA expression, processing and/or function. The circRNA inhibitor of the present invention is a compound that inhibits or reduces the activity or expression of circLTBP2. The term “inhibiting circLTBP2 expression” means that the production of circLTBP2 in target cells after treatment is less than the amount produced prior to treatment or neutralize the activity of existent amount. One skilled in the art can readily determine whether circLTBP2 expression has been inhibited in the target cells, using for example the techniques for determining miRNA transcript level. In some embodiments, the circRNA inhibitor of the present invention is a compound such as nucleic acid that hybridizes with circLTBP2 or having sequence complementarity to that of circLTBP2. In some embodiments, circRNA inhibitor of the present invention is a compound such as nucleic acid having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 9899 or 100% sequence complementarity to that of circLTBP2. Suitable circRNA inhibitors include double-stranded RNA (such as short- or small-interfering RNA or "siRNA"), antisense nucleic acids, and enzymatic RNA molecules such as ribozymes. Each of these compounds can be targeted to a given circRNA and destroy or induce the destruction of the target circRNA.
Suitable circRNA inhibitors include double-stranded RNA (such as short- or small-interfering RNA or "siRNA"), antagomirs, antisense nucleic acids, and enzymatic RNA molecules such as ribozymes. Each of these compounds can be targeted to a given miRNA and destroy or induce the destruction of the target miRNA. For example, expression of a given miRNA can be inhibited by inducing RNA interference of the miRNA with an isolated double-stranded RNA ("dsRNA") molecule which has at least 90%, for example 90%; 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, sequence homology with at least a portion of the miRNA. In some embodiments, the dsRNA molecule is a "short or small interfering RNA" or "siRNA". siRNA useful in the present methods comprise short double-stranded RNA from about 17 nucleotides to about 29 nucleotides in length, preferably from about 19 to about 25 nucleotides in length. The siRNA comprise a sense RNA strand and a complementary antisense RNA strand annealed together by standard Watson-Crick base-pairing interactions (hereinafter "base-paired"). The sense strand comprises a nucleic acid sequence which is substantially identical to a nucleic acid sequence contained within the target miRNA. As used herein, a nucleic acid sequence in a siRNA which is "substantially identical" to a target sequence contained within the target mRNA is a nucleic acid sequence that is identical to the target sequence, or that differs from the target sequence by one or two nucleotides. The sense and antisense strands of the siRNA can comprise two complementary, single-stranded RNA molecules, or can comprise a single molecule in which two complementary portions are base- paired and are covalently linked by a single-stranded "hairpin" area. The siRNA can also be altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non- nucleotide material, such as to the end(s) of the siRNA or to one or more internal nucleotides of the siRNA, or modifications that make the siRNA resistant to nuclease digestion, or the substitution of one or more nucleotides in the siRNA with deoxyribonucleotides. One or both strands of the siRNA can also comprise a 3 overhang. As used herein, a "3' overhang" refers to at least one unpaired nucleotide extending from the 3'-end of a duplexed RNA strand. Thus, in some embodiments, the siRNA comprises at least one 3' overhang of 1 to about 6 nucleotides (which includes ribonucleotides or deoxyribonucleotides) in length, preferably from 1 to about 5 nucleotides in length, more preferably from 1 to about 4 nucleotides in length, and particularly preferably from about 2 to about 4 nucleotides in length. In some embodiments, the 3' overhang is present on both strands of the siRNA, and is 2 nucleotides in length. For example, each strand of the siRNA can comprise 3' overhangs of dithymidylic acid ("TT") or diuridylic acid ("uu").
The siRNA can be produced chemically or biologically, or can be expressed from a recombinant plasmid or viral vector, as described above. Exemplary methods for producing and testing dsRNA or siRNA molecules are described in U.S. published patent application 2002/0173478 to Gewirtz and in U.S. published patent application 2004/0018176 to Reich et al., the entire disclosures of which are herein incorporated by reference. In some embodiments, the circRNA of the present invention is an antisense nucleic acid. As used herein, an "antisense nucleic acid" refers to a nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-peptide nucleic acid interactions, which alters the activity of the target RNA. Antisense nucleic acids suitable for use in the present methods are single-stranded nucleic acids (e.g., RNA, DNA, RNA-DNA chimeras, PNA) that generally comprise a nucleic acid sequence complementary to a contiguous nucleic acid sequence in a miRNA. Preferably, the antisense nucleic acid comprises a nucleic acid sequence that is 50-100% complementary, more preferably 75-100% complementary, and most preferably 95-100% complementary to a contiguous nucleic acid sequence in the circRNA. In some embodiments, the antisense nucleic acid activates RNase H or some other cellular nuclease that digests the circRNA/antisense nucleic acid duplex and thus inhibits the expression of the targeted circRNA. In some embodiments, the antisense binds to the miR-338-3p target site in the circLTBP2 nucleic acid sequence. This has the effect of blocking said target site (for example, by steric interference), preventing its recognition and binding by miR-338-3p, and thus inhibiting the sequestration of miR-338-3p by circLTBP2. Thus the binding of the nucleic acid antisense to the miR-338-3p mRNA target site does not induce gene silencing (e.g. by circRNA degradation or translational repression) of said target circRNA. Binding of the antisense to a miR-338-3p mRNA target site may occur via complementary base pairing, as described above. Thus, in some embodiments, binding between the antisense and the miR-338-3p mRNA target site occurs via complementary base pairing between at least one nucleotide present in the antisense and a corresponding nucleotide present in the miR-338-3p mRNA target site, such that at least a portion of the antisense and the miR-338-3p mRNA target site together define a base-paired nucleic acid duplex. Said complementary base pairing (and thus duplex formation) can occur over a region of two or more contiguous nucleotides of the miR-338-3p mRNA target site (e.g. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides). A
base- paired nucleic acid duplex formed when the antisense binds to the miR-338-3p mRNA target (as described above) may comprise one or more mismatch pairings. In some embodiments, two or more regions of complementary base-paired nucleic acid duplex (e.g.3, 4, 5 or 6) are formed, wherein each region is separated from the next by one or more mismatch pairings. Antisense nucleic acids can also contain modifications of the nucleic acid backbone or of the sugar and base moieties (or their equivalent) to enhance target specificity, nuclease resistance, delivery or other properties related to efficacy of the molecule. Such modifications include cholesterol moieties, duplex intercalators such as acridine or the inclusion of one or more nuclease-resistant groups. Antisense nucleic acids can be produced chemically or biologically, or can be expressed from a recombinant plasmid or viral vector, as described below. Exemplary methods for producing and testing are within the skill in the art; see, e.g., Stein and Cheng (1993), Science 261:1004 and U.S. Pat. No. 5,849,902 to Woolf et al., the entire disclosures of which are herein incorporated by reference. In some embodiments, the circRNA inhibitor of the present invention is an endonuclease. In some embodiments, the endonuclease is CRISPR-cas. In some embodiment, the endonuclease is CRISPR-cas13 (“cas13”). Like Cas9, Cas13 uses a guide RNA (CRISPR-RNA, aka crRNA) to identify its substrate, which is RNA rather than DNA. There are currently four subtypes identified in the Cas13 family, including Cas13a (aka C2c2), Cas13b, Cas13c, and Cas13d. All Cas13 enzymes require a 60–66-nucleotide-long crRNA to ensure target specificity. Similar to the gRNA in the CRISPR/Cas9 system, the crRNA used by Cas13 forms a short hairpin structure next to a short spacer sequence (28–30 nucleotides) that is specific to the target transcript. Since CRISPR/Cas13 mediates RNA degradation, it holds the promise to replace or complement RNA interference (RNAi) approaches or other systems that interfere with transcript levels, such as CRISPRi. The circRNA inhibitor of the present invention can be obtained using a number of standard techniques. For example, the circRNA inhibitor of the present invention can be chemically synthesized or recombinantly produced using methods known in the art. Typically, the circRNA inhibitors of the present invention are chemically synthesized using appropriately protected
ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. Commercial suppliers of synthetic RNA molecules or synthesis reagents include, e.g., Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part of Perbio Science, Rockford, 111., USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA) and Cruachem (Glasgow, UK). In some embodiments, the circRNA inhibitor of the present invention is resistant to degradation by nucleases. One skilled in the art can readily synthesize nucleic acids which are nuclease resistant, for example by incorporating one or more ribonucleotides that are modified at the 2'- position into the miRNAs. Suitable 2'-modified ribonucleotides include those modified at the 2'-position with fluoro, amino, alkyl, alkoxy, and O-allyl. Alternatively, the circRNA inhibitor of the present invention can be expressed from recombinant linear or circular DNA plasmids using any suitable promoter. Suitable promoters for expressing RNA from a plasmid include, e.g., the U6 promoter sequence, or the cytomegalovirus promoters. Selection of other suitable promoters is within the skill in the art. The recombinant plasmids of the present invention can also comprise inducible or regulatable promoters for expression of the circRNA inhibitor of the present invention in target cells. The circRNA inhibitor of the present invention that is expressed from recombinant plasmids can be isolated from cultured cell expression systems by standard techniques. The circRNA inhibitor of the present invention which is expressed from recombinant plasmids can also be delivered to, and expressed directly in, target cells. The use of recombinant plasmids to deliver the circRNA inhibitor of the present invention to target cells is discussed in more detail below. The circRNA inhibitor of the present invention can be expressed from a separate recombinant plasmid, or can be expressed from a unique recombinant plasmid. Preferably, the circRNA inhibitor of the present invention is expressed as the nucleic acid precursor molecules from a single plasmid, and the precursor molecules are processed into the functional circRNA inhibitor by a suitable processing system, including processing systems extant within target cells. Other suitable processing systems include, e.g., the in vitro Drosophila cell lysate system as described in U.S. published application 2002/0086356 to Tuschl et al. and the E. coli RNAse III system described in U.S. published patent application 2004/0014113 to Yang et al., the entire disclosures of which are herein incorporated by reference.
Selection of plasmids suitable for expressing the circRNA inhibitor of the present invention, methods for inserting nucleic acid sequences into the plasmid to express the gene products, and methods of delivering the recombinant plasmid to the cells of interest are within the skill in the art. See, for example, Zeng et al. (2002), Molecular Cell 9:1327-1333; Tuschl (2002), Nat. Biotechnol, 20:446-448; Brummelkamp et al. (2002), Science 296:550-553; Miyagishi et al. (2002), Nat. Biotechnol.20:497-500; Paddison et al. (2002), Genes Dev.16:948-958; Lee et al. (2002), Nat. Biotechnol.20:500-505; and Paul et al. (2002), Nat. Biotechnol.20:505-508, the entire disclosures of which are herein incorporated by reference. In some embodiments, a plasmid expressing the circRNA inhibitor of the present invention comprises a sequence encoding a circRNA inhibitor precursor under the control of the CMV intermediate early promoter. As used herein, "under the control" of a promoter means that the nucleic acid sequences are located 3' of the promoter, so that the promoter can initiate transcription of the circRNA inhibitor coding sequences. The circRNA inhibitor of the present invention can also be expressed from recombinant viral vectors. It is contemplated that the circRNA inhibitor of the present invention can be expressed from separate recombinant viral vectors, or from a unique viral vector. The circRNA inhibitor expressed from the recombinant viral vectors either can be isolated from cultured cell expression systems by standard techniques or can be expressed directly in target cells. The use of recombinant viral vectors to deliver the circRNA inhibitor to target cells is discussed in more detail below. The recombinant viral vectors of the present invention comprise sequences encoding the circRNA inhibitor compound of the present invention and any suitable promoter for expressing the circRNA inhibitor sequences. Suitable promoters include, for example, the U6 or HI RNA pol III promoter sequences, or the cytomegalovirus promoters. Selection of other suitable promoters is within the skill in the art. The recombinant viral vectors of the present invention can also comprise inducible or regulatable promoters for expression of the circRNA inhibitor in target cells. Any viral vector capable of accepting the coding sequences for The circRNA inhibitor of the present invention can be used; for example, vectors derived from adenovirus (AV); adenoassociated virus (t); retroviruses (e.g., lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like. The tropism of the viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate.
For example, lentiviral vectors of the present invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors of the present invention can be made to target different cells by engineering the vectors to express different capsid protein serotypes. For example, an AAV vector expressing a serotype 2 capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector. Techniques for constructing AAV vectors which express different capsid protein serotypes are within the skill in the art; see, e.g., Rabinowitz J. E. et al. (2002), J Virol 76:791801, the entire disclosure of which is herein incorporated by reference. The circRNA inhibitor can be administered to a patient by any means suitable for delivering these compounds to target cells. For example, the circRNA inhibitor can be administered by methods suitable to transfect cells of the patient with these compounds, or with nucleic acids comprising sequences encoding these compounds. Preferably, the cells are transfected with a plasmid or viral vector comprising sequences encoding at least one circRNA inhibitor. The circRNA inhibitor can be administered to a patient by any suitable enteral or parenteral administration route. Suitable enteral administration routes for the present methods include, e.g., oral, rectal, or intranasal delivery. Suitable parenteral administration routes include, e.g., intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra- arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue injection (e.g., intra-retinal injection, or subretinal injection); subcutaneous injection or deposition, including subcutaneous infusion (such as by osmotic pumps); direct application to the tissue of interest, for example by a catheter or other placement device (e.g., an implant comprising a porous, non-porous, or gelatinous material); and inhalation. Preferred administration routes are injection, infusion and direct injection into the tumor tissue. One skilled in the art can readily determine a therapeutically effective amount of the circRNA inhibitor to be administered to a given patient, by taking into account factors such as the size and weight of the patient; the extent of disease penetration; the age, health and sex of the patient; the route of administration; and whether the administration is regional or systemic. An effective amount of said compound can be based on the approximate or estimated body weight of a patient to be treated. Preferably, such effective amounts are administered parenterally or enterally, as described herein. For example, an effective amount of the compound administered
to a patient can range from about 5-10000 micrograms/kg of body weight and is preferably between about 5-3000 micrograms/kg of body weight, and is preferably between about 700- 1000 micrograms/kg of body weight, and is more preferably greater than about 1000 micrograms/kg of body weight. One skilled in the art can also readily determine an appropriate dosage regimen for the administration of the compound to a given patient. For example, the compound can be administered to the patient once (e.g., as a single injection or deposition). In some embodiments, the circRNA inhibitor of the present invention is administered to the patient in combination with one or more TGF-beta inhibitor(s). As used herein, the term "TGF- beta" has its general meaning in the art and refers to one or more members of the transforming growth factor-beta family of proteins, e.g., TGF-beta 1 , TGF- beta 2, and TGF- beta 3. As used herein, the term "TGF-beta inhibitor" refers to an agent having the ability to directly or indirectly inhibit a biological function of TGF-beta. Thus, TGF-beta inhibitors include, but are not limited to, inhibitors (e.g., blocking (neutralizing) antibodies) specific for TGF-beta, soluble TGF-beta receptors (which would competitively inhibit TGF-beta), membrane-bound TGF- beta receptors, protease inhibitors that inactivate a protease responsible for activating a precursor TGF-beta into mature TGF-beta, inhibitors (e.g., antibodies or small molecules) specific to TGF-beta receptors (Types I, II or III) that prevent TGF-beta binding to the receptor, siRNA or antisense RNA that block expression of TGF-beta or combinations of the foregoing. In some embodiments, the TGF-beta inhibitor is fresolimumab. The circRNA inhibitors of the present invention are preferably formulated as pharmaceutical compositions, prior to administering to a patient, according to techniques known in the art. Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen-free. As used herein, "pharmaceutical formulations" include formulations for human and veterinary use. Methods for preparing pharmaceutical compositions of the present invention are within the skill in the art, for example as described in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), the entire disclosure of which is herein incorporated by reference. The present pharmaceutical formulations comprise circRNA inhibitor (e.g., 0.1 to 90% by weight), or a physiologically acceptable salt thereof, mixed with a pharmaceutically-acceptable carrier. The pharmaceutical formulations of the present invention can also comprise circRNA inhibitor which are encapsulated by liposomes and a pharmaceutically-acceptable carrier. Preferred pharmaceutically-acceptable carriers are water, buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.
Pharmaceutical compositions of the present invention can also comprise conventional pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents. Suitable additives include, e.g., physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (such as, for example, calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). Pharmaceutical compositions of the present invention can be packaged for use in liquid form or can be lyophilized. Use miR-338-3p mimic: A further object of the present invention relates to a method of treating an intrahepatic cholangiocarcinoma in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a miR-338-3p mimic. As used herein, the term “miRNA mimic” refers to synthetic small non-coding RNAs capable of entering the RNAi pathway and regulating gene expression. As used herein, the term “synthetic miRNA” refers to any type of miRNA sequence, other than an endogenous miRNA. miRNA mimics imitate the function of endogenous miRNAs and can be designed as mature, double stranded (duplex) molecules or mimic precursors e.g., pri-miRNAs or pre-miRNAs. The miRNA mimic may comprise an effector sequence which is substantially identical to the effector sequence of the corresponding endogenous miRNA. miRNA mimics can be comprised of modified and/or unmodified RNA, DNA, RNA-DNA hybrids or alternative nucleic acid chemistries. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES: Figure 1. CircLTBP2, a TGFβ-induced circRNA, predicts a poor prognosis in iCCA patients. (A) CircLTBP2 (hsa_circ_0032603) relative expression in 20 freshly frozen iCCA tumors from a French national cohort (separated in two groups: circLTBP2low and
circLTBP2high, according to their circLTBP2 median expression). (B) Kaplan-Meier plots and log-rank statistics analysis revealed a significant decreased overall survival (OS) and relapse free survival (RFS) for iCCA patients with a high expression of circLTBP2. (C) CircLTBP2 relative expression in hepatocellular carcinoma (HCC) and CCA tumors unraveled circLTBP2 specificity for CCA. (D) Kaplan-Meier plots and log-rank statistics analysis endorsed the association of circLTBP2 expression with OS in the independent cohort of CCA while no correlation was found in HCC. Data are presented as means ±SD. (* P-value < 0.05 ** P- value < 0.01, *** P-value < 0.001, n ≥ 3). Abbreviations: iCCA (intrahepatic cholangiocarcinoma). Figure 2. Overexpression of circLTBP2 promotes iCCA cell proliferation, migration, and resistance to gemcitabine-induced apoptosis in vitro and tumor growth in vivo. (A) Relative confluency of cultured HuCC-T1 cells was evaluated for 132 hours (n=3 per group). HuCC-T1 cells overexpressing circLTBP2 showed an increased proliferation. (B) Cell migration was evaluated by wound healing assay performed on HuCC-T1 cells overexpressing circLTBP2 compared to the empty vector control group (n=9 per group). HuCC-T1 cells were cultured in low FBS concentration medium (1% FBS) and with Mitomycin C (10 µg. mL-1) to inhibit cell proliferation. (C) HuCC-T1 cells resistance to gemcitabine-induced apoptosis (10 ng/mL) was evaluated every 4 hours using a red fluorescent Annexin V labelling, in both the circLTBP2 overexpressing and control groups. Mouse subcutaneous tumor model was used to evaluate the impact of circLTBP2 overexpression on tumor growth (D) and volume (E).5 mice were included in the control group and 6 mice were included in the circLTBP2 overexpression group. Statistical analyses for (E) were performed by a Mann–Whitney test. Statistical analyses for (A), (B), (C) and (D) were performed by a 2-way-Anova test (* P-value < 0.05 ** P-value < 0.01, *** P-value < 0.001, n ≥ 3). Abbreviations: pLV-circLTBP2 (circLTBP2 overexpression vector), pLV-circRNA (empty vector), TGFβ (Transforming-Growth Factor β). Figure 3. miR-338-3p, a known tumor suppressor miRNA predicts a good prognosis in iCCA patients.. (A) Mir-338-3p relative expression in 20 freshly frozen iCCA tumors from a French national cohort (separated in two groups: miR-338-3plow and miR-338-3phigh, according to their circLTBP2 median expression). (B) Kaplan-Meier plots and log-rank statistics analysis revealed a significant decreased overall survival (OS) and relapse free survival (RFS) for iCCA patients with a low expression of miR-338-3p. Statistical analyses for (B) were performed by a Mann–Whitney test. (* P-value < 0.05 ** P-value < 0.01, *** P-value < 0.001, n ≥ 3).
Abbreviations: GSEA (Gene Set Enrichment Analysis), NES (Normal Enrichment Score), TGFβ (Transforming-Growth Factor β). Figure 4. The restoration of miR-338-3p levels in iCCA cells reversed the pro-tumorigenic effects driven by circLTBP2. (A) Relative levels of miR-338-3p in HuCC-T1 were assessed by q-RT-PCR in cells overexpressing circLTBP2 vs. in cells transfected with the empty vector. In addition, miR-338-3p mimics (5nM) were added with the aim of rescuing the sponging mediated by circLTBP2. (B) Relative confluency of cultured HuCC-T1 cells was evaluated for 96 hours in HuCC-T1 cells overexpressing circLTBP2 compared to the empty vector control group (n=3 per group). In addition, miR-338-3p mimics were added aiming at restoring the phenotype. (C) Cell migration was evaluated by wound healing assay performed on HuCC-T1 cells overexpressing circLTBP2 compared to the empty vector control group (n=3 per group). In addition, miR-338-3p mimics (5nM) were added aiming at restoring the phenotype. (D) HuCC-T1 cells resistance to gemcitabine-induced apoptosis (10 ng/mL) was evaluated every 4 hours using a red fluorescent Annexin V labelling, in both the circLTBP2 overexpressing and control groups. In addition, miR-338-3p mimics were added aiming at restoring the phenotype (5nM). Statistical analyses for (A) were performed by a Mann–Whitney test. Statistical analyses for (B), (C) and (D) were performed by a 2-way-Anova test (* P-value < 0.05 ** P-value < 0.01, *** P-value < 0.001, n ≥ 3). Abbreviations: pLV-circLTBP2 (circLTBP2 overexpression vector), pLV-circRNA (empty vector). Figure 5. Circulating vesicles containing circLTBP2 in serum are associated with poor OS in iCCA patients. (A) Quantification by q-RT-PCR of EV-embed circLTBP2 in sera from metastatic advanced iCCA patients (Rennes cohort, n=104). Sera from healthy individuals were used as control (Établissement français du sang n=12). (B) Positive correlation of circLTBP2 expression in matched tissues and serum samples (patients for which both samples are available, n=17) (C) EV-embed circLTBP2 relative expression in sera from iCCA patients whose survival data were available (separated in two groups: circLTBP2low and circLTBP2high, according to their circLTBP2 median expression). (D) Kaplan-Meier plots and log-rank statistics analysis revealed a significant decreased overall survival (OS) for iCCA patients with a high expression of vesicles-embed circLTBP2. Levels of circLTBP2 were not associated with the relapse-free survival (RFS). Abbreviations: pLV-circLTBP2 (circLTBP2 overexpression vector), pLV- circRNA (empty vector).
EXAMPLE: Material & Methods Patients and tissue samples Freshly frozen human CCA were obtained from the National Liver Network (BB-0033-00085). A validating cohort, which included 12 iCCA, 5 dCCA, 2 pCCA and 20 HCC, was also used. Serum samples were obtained from patients with advanced metastatic iCCA (n= 104) from the CLCC Eugène Marquis (Rennes, France). Cell lines and functional assays HuCC-T1 (RCB-1960) and HuH-28 (RCB-1943) CCA cell lines were obtained from the RIKEN BioResource Center in Japan. SG231, Mz-ChA-1, TFK-1 and SK-ChA-1 were kindly provided by Dr. Laura Fouassier from the Centre de Recherche Saint-Antoine (Inserm U938, Paris, France). Culture conditions are described in the supplementary materials and methods. Full length circLTBP2 (hsa-circ-0032603) was cloned into pLV-circ_GFP by Creative biogene (NY, USA) to perform gain of function experiments. Antisense LNA GapmeRs that target the circularization junction of circLTBP2 (#339511, Qiagen) were used for loss of function experiments. Cell proliferation, migration and apoptosis assays were performed using an Incucyte S3 device (Sartorius, USA). CircRNA microarray analysis Total RNA, including circRNAs, was purified with an miRNeasy kit (Qiagen, 217004). Arraystar Human circRNA Microarray v2.0 (Arraystar, Rockville, MD, USA) were used to screen for novel TGFβ-regulated circRNAs in human CCA cell lines. q-RT-PCR, western blot and immunoprecipitation (IP) Quantitative reverse-transcription PCR (q-RT-PCR) and western blot were performed as previously described. [13] Primers and antibodies are listed in Table S1. MiRNA IP was carried out according to the manufacturer’s instructions (miRNA Target IP kit #25500, Active Motif, Inc). Briefly, miR-338-3p mimics (miRCURY LNA miRNA Mimic, Qiagen, 5nM) were transfected into HuCC-T1 cells for 24 hours. The IP was performed using a pan-Ago antibody to identify miRNA/mRNA and miRNA/circRNA complexes. circRNA pulldown assay
A circLTBP2 pulldown assay was carried out by Creative Biogene (NY, USA). Briefly, a labeled circRNA expression vector was generated and co-transfected into HuCC-T1 cells with a capture protein expression vector. Afterwards, a pulldown assay was done by using a labeled antibody that binds to the induced capture protein, which allows for the pulldown of circLTBP2-miRNA complexes. Eluted miRNA were identified by miRNA-sequencing. Tumor xenograft model HuCC-T1 cells were modified to overexpress either an empty vector (pLV-circRNA-GFP) or circLTBP2 (pLV-circLTBP2-GFP) and were infected with GL261-Luc (CMV-Firefly luciferase lentivirus (Neo), PLV-10064-50, Cellomics Technology, USA).4,000,000 cells were implanted on the flanks of 8-week-old female NSG mice (Charles River, USA). All animal procedures followed the European Community Directive guidelines (Agreement B35- 639238- 40, Biosit Rennes, France; DIR #7163) and were approved by the local ethics committee. Tumor growth was evaluated by measuring the size of the tumor with caliper. 90 days after implantation, mice were sacrificed and the tumors, liver, and lung tissues were isolated and analyzed at the molecular level. The presence of metastases in the liver and in the lungs was evaluated by bioluminescence. Statistical analyses All statistical analyses were conducted using Prism 8 software (from GraphPad Software) and the results were presented as mean values ± standard deviation or median values. Comparison between variables was made using either Student’s t-test or Mann-Whitney U test. For multiple sets of multivariate comparisons, two-way analysis of variance was applied. Survival curves were analyzed through Kaplan-Meier method and log-rank test. A P value < 0.05 was considered statistically significant. Results: HuCC-T1 is a TGFβ-responsive iCCA cell line CCA is a rare cancer with limited characterized in vitro models [14]. Therefore, we first characterized 3 iCCA (HuCC-T1, HuH-28, SG231) and 3 eCCA (Mz-ChA-1, TFK-1, SK-ChA- 1) cell lines. Mz-ChA1 and TFK-1 displayed an epithelial phenotype (high expression of E- Cadherin, CDH1 and no expression of Vimentin, VIM) while HuH-28 displayed a mesenchymal phenotype (high VIM / no CDH1). SK-ChA-1, SG231 and HuCC-T1 exhibited an intermediate phenotype with expression of both CDH1 and VIM (data not shown). Next,
we evaluated their response to TGFβ by measuring the level of phosphorylated SMAD3 (data not shown) and the expression of TGFβ target genes (SERPINE1, SMAD7, BIRC3). HuCC- T1 and HuH-28 cell lines were fully responsive to TGFβ, with a significant SMAD3 phosphorylation, up-regulation of SERPINE1, SMAD7 and down-regulation of BIRC3. In addition, these effects were reversed in presence of galunisertib (LY2157299), a selective inhibitor of TGFBR1 (data not shown). Subsequent experiments were performed in HuCC-T1 cells, which exhibited better culture conditions and transfection efficiency. CircLTBP2 is a TGFβ-induced circRNA predictive of poor prognosis in iCCA Gene expression profiling using Arraystar microarrays identified 119 circRNAs significantly (P<0.001 and FC>2) deregulated by TGFβ (1 ng/mL, 16 hours) in HuCC-T1, including 85 up- and 34 down-regulated circRNAs (data not shown). Interestingly, more than 50% of circRNAs and their linear counterparts were co-regulated in response to TGFβ (data not shown). Out of the 119 TGFβ-regulated circRNAs, 43 were predicted to act as miRNA sponges data not shown). A gene ontology (GO) analysis revealed that these circRNAs were associated to biological processes regulated by TGFβ, such as cell migration and apoptosis (data not shown). The expression of 5 randomly selected TGFβ-induced circRNAs was validated by q-RT-PCR (data not shown). Next, we evaluated their clinical relevance in resected iCCA tumors (cohort 1, n=20). Among the 5 candidates, circLTBP2 was associated with a poor prognosis since its expression was predictive of a significant reduced overall (OS) and relapse-free (RFS) survival (P<0.01) (Fig.1A-B). The association of circLTBP2 expression and OS was further validated in two additional cohorts of liver cancers, including hepatocellular carcinoma (HCC) and CCA (Fig.1D). Interestingly, the expression of circLTBP2 was very low in HCC and not associated with OS (Fig.1C-D). CircLTBP2 was detected in both intrahepatic and extrahepatic CCA tissues and induced by TGFβ in all CCA cell lines with the exception of SG231 (data not shown). CircLTBP2 is a large exonic circRNA (2165 bases) composed of exons 2 to 16 of LTBP2 mRNA. Its circular structure was confirmed by Sanger sequencing (data not shown). We developed sets of convergent and divergent primers to specifically detect the circLTBP2 isoform hsa-circ-0032603 and its linear LTBP2 counterpart (data not shown). In HuCC-T1 cells, circLTBP2 induction by TGFβ was the highest (data not shown) and abolished in presence of galunisertib or SMAD3 inhibitor SIS3 (data not shown).
CircLTBP2 exerts pro-oncogenic actions in iCCA First, we performed gain-of-function (GOF) experiments by infecting HuCC-T1 cells with a vector that contained circLTBP2 (pLV-circLTBP2) (data not shown). Overexpression was validated in several HuCC-T1 clones and 3 of them demonstrating similar levels of circLTBP2 expression as seen after TGFβ treatment were selected for functional assays. Importantly, the expression of linear LTBP2 was not affected by circLTBP2 overexpression (data not shown). TGFβ contributes to CCA progression by stimulating cell proliferation, migration and by inhibiting apoptosis. [15] Thus, we investigated the impact of circLTBP2 on these processes using an Incucyte S3 device. HuCC-T1 cells stably overexpressing circLTBP2 exhibited significantly higher proliferation and migration abilities when compared to cells infected with an empty vector (Fig. 2A-B). In addition, circLTBP2 overexpression promoted resistance to gemcitabine-induced apoptosis (Fig. 2C). The pro-tumorigenic potential of circLTBP2 was further evaluated in vivo by injecting genetically engineered HuCC-T1 cells into NSG mice (data not shown). Tumors developed in mice injected with cells overexpressing circLTBP2 were significantly bigger in size (856±336 vs 1478±292.07 mm3, P<0.001) and weight (0.6±021 vs 1.12±0.41 g, P<0.05) (Fig. 2D-E). CircLTBP2 overexpression in the resected tumors was confirmed by q-RT-PCR (data not shown). No difference was observed in the number or size of lung metastases (data not shown). Next, loss-of-function (LOF) experiments using LNA GapmeRs specifically targeting circLTBP2 circularization junction were performed (data not shown). Importantly, these GapmeRs were shown to specifically decrease the expression of circLTBP2 without impacting the expression of its linear counterpart (data not shown). Validating our previous observation, circLTBP2 LOF in HuCC-T1 cells resulted in a decrease of cell proliferation and migration, and an improved sensitivity to gemcitabine (data not shown). CircLTBP2 acts as a sponge for miR-338-3p, a tumor suppressor miRNA To gain insight into the molecular mechanisms driving the effects circLTBP2, we performed a circLTBP2 pull-down assay followed by miRNA sequencing. Due to the large size of circLTBP2 and the diverse functional impact of circLTBP2, we hypothesized that circLTBP2 may act as a sponge for tumor suppressor miRNAs. A decision-making pattern was established to select the most promising miRNA candidates (data not shown). By circRNA pull-down, we identified 94 miRNAs possibly interacting with circLTBP2, among them 69 were recorded in databases. The selection of miRNAs also relied on a strong dependency to circLTBP2, as determined by the number of predicted binding sites (>2) and the enrichment score (>1.3) of
the predicted miRNA targets in the gene expression profile of HuCC-T1 cells stimulated by TGFβ. In total, 8 miRNAs met the selection criteria (miR-338-3p, miR-3064-5p, miR1270, miR-1294, miR-34c-5p, miR324-3p, miR-1914-3p, miR-34a-5p) (data not shown). Among them, miR-338-3p had the highest number of predicted binding sites (n=7) on circLTBP2 (Fig. 3B), and the highest enrichment score (NES=1.86, P<0.01), as determined by GSEA (data not shown). To confirm the pull-down assay, we performed a miRNA IP assay that demonstrated the direct interaction between miR-338-3p and circLTBP2 (data not shown). Among 16 miR- 338-3p targets possibly involved in biological processes regulated by circLTBP2, the expression of 10 was significantly increased by TGFβ (data not shown). Four of these targets (SOX4, ANHAK2, FURIN, NET1) also exhibited a small but significant decrease in expression in cells transfected with miR-338-3p mimics (data not shown). Mirroring the results observed with circLTBP2 (Fig. 1), a reduced expression of miR-338-3p in human CCA tumors was associated with a significant reduced overall (OS) and relapse-free (RFS) survival (P<0.01) (Fig. 3A-B). Next, in order to determine if the pro-tumorigenic effects driven by circLTBP2 resulted from the sequestration of miR-338-3p, we conducted rescue experiments using miRNA mimics. Importantly, circLTBP2 expression does not affect the expression of miR-338-3p (Fig. 4A). Rescuing miR-338-3p levels was able to decrease circLTBP2-induced cell migration and to sensitize cells to gemcitabine-induced apoptosis (Fig.4B-D), demonstrating that circLTBP2 contributes to gemcitabine resistance at least by sponging miR-338-3p. Circulating circLTBP2 is associated with poor OS in iCCA patients Finally, we aimed at evaluating the clinical relevance of circLTBP2 as a prognostic biomarker for iCCA, easily detectable by non-invasive methods. Thus, we first determined whether circLTBP2 could be embedded into extracellular vesicles (EVs) and secreted. EVs (mainly exosomes) were isolated by ultracentrifugation and qualified by qNano, transmission electron microscopy and expression of specific surface markers (data not shown). Interestingly, circLTBP2 was expressed in EVs secreted by HuCC-T1 cells and its expression in EVs was greater in cells pre-treated by TGFβ (data not shown). In addition, circLTBP2 was more stable than its linear counterpart with a half-life greater than 24 hours (data not shown). More interestingly, the level of circLTBP2 was significantly increased in EVs isolated from the serum of patients with advanced, metastatic iCCA (n=103) vs healthy individuals (Fig. 5A). A significant correlation between the levels of circLTBP2 in the tumor tissues and in the serum was also highlighted (Fig.5B). From patients with available survival data (n=39) we generated two groups based on the median expression of circLTBP2 (Fig. 5C). Statistical analysis
demonstrated that a high expression of circulating circLTBP2 was significantly associated with a reduced OS (Fig.5D). Discussion: In this study, we identified a novel signature of circRNAs modulated by TGFβ in iCCA. In addition, we characterized the role and the underlying molecular mechanisms by which TGFβ- induced circLTBP2 modulates iCCA progression. We demonstrated that an increased expression of circLTBP2 promotes iCCA cell proliferation and migration, as well as resistance to gemcitabine-induced apoptosis. Mechanistically, circLTBP2 exerts pro-oncogenic actions by modulating the activity of miR-338-3p, a well-known tumor suppressor miRNA. [16] We also report that circLTBP2 expression in tissues and sera was associated with a worse prognosis. Latent-TGFβ binding protein 2 (LTBP2) belong to the family of LTPB proteins involved notably in the bioavailability of TGFβ at the cell surface. However, LTBP-2 is unique in the family as it does not bind to latent TGFβ. LTBP2 associates with ECM and it has been suggested that it may indirectly regulate the activation of TGFβ by releasing LTBP-1 from microfibrils. [17] Increased expression of LTBP2 has been reported in several cancers and associated with tumor progression, notably in HCC. [18] Here, we provide the first evidence that the induction of its circular counterpart participates to CCA progression by sponging miR-338-3p and thus protecting its pro-oncogenic targets from degradation. This miRNA plays a crucial role in tumor progression and its activity is tightly regulated in cancer. Many pathways depend on its activity, including WNT, MAPK, and PI3K/AKT, all of which are involved in the progression of iCCA. [19] Accordingly, miR-338-3p controls numerous hallmarks of cancer cells, such as inhibiting cell proliferation signals, inducing cell death, or decreasing angiogenesis. [16] MiR-338-3p has also a strong prognostic value for biliary tract cancers. Low levels of miR-338-3p have been found in several advanced stage (TNM3) biliary tract tumors associated with significant lymph node infiltration. MiR-338-3p has also been identified in a signature of 3 miRNAs that predict survival in patients with iCCA. Patients with low levels of miR-338-3p had the worse OS and RFS. [20] Our data suggest that miR-338-3p possibly targets SOX4, a key gene involved in epithelial-to-mesenchymal transition (EMT), modulating master regulators TWIST1, SNAI1 and ZEB1. [21] The regulation of SOX4 by miR-338-3p has already been described in several cancers (e.g. breast cancer, kidney cancer) and associated with cell proliferation and migration. [22] In lung cancer, miR-338-3p has been shown to suppress metastasis by targeting SOX4. [23]
Only few studies have reported in depth functional studies of circRNAs driving iCCA oncogenesis so far. [8, 9] For example, circACTN4 acts as a signaling nexus allowing for the coordinated activation of Hippo and Wnt pathways in iCCA. Mechanistically, circACTN4 is able to sponge miR-424-5p in the cytoplasm allowing for the expression of Yes-associated protein 1 (YAP1) and to recruit YBX1 at the promoter of FZD7 in the nucleus. [10, 12]. CircRNA MBOAT2 promotes iCCA progression and lipid metabolism reprogramming by stabilizing PTBP1 to facilitate FASN mRNA cytoplasmic export. [24] Interestingly, some circRNAs could be translated. Thus, the protein encoded by IL6-induced circGGNBP2 was shown to promote CCA cell growth and metastasis, notably through a positive regulatory loop modulating IL-6/STAT3 signaling. [25] Secreted circRNAs could also play a key role in CCA carcinogenesis, as illustrated by circ_0020256 in exosomes from tumor-associated M2 macrophages which has been shown to promote CCA cell proliferation, migration, and invasion by modulating a miR-432-5p/E2F3 axis. [26] Although further validation using large cohorts of patients will be needed to confirm the link between circLTBP2 expression and survival, our data highlight the clinical relevance of circLTBP2 as a prognostic biomarker in three small but independent cohorts of iCCA and HCC. The diagnosis of some iCCA may represent a clinical challenge due to anatomo-morphological similarities between tumors developing along the hepatobiliary tree. [27] In this study, we report a differential expression of circLTBP2 between iCCA and HCC, suggesting that circLTBP2 could be used as a biomarker helping to differentiate these two types of tumors. CCA comprises a heterogeneous group of tumors and applying a systematic and effective therapeutic approach for all patients is challenging. The current first-line reference treatment is a combination of gemcitabine and cisplatin. However, the benefit of this treatment is modest, and iCCA remains a poor prognosis cancer. [28-30] Our data further suggest that inhibiting circLTBP2 (e.g. by using antisense oligonucleotides) could be relevant to sensitize iCCA cells to gemcitabine. Translational studies supporting this point will be required. In summary, our study demonstrates that circRNAs modulate iCCA carcinogenesis and may serve as clinically relevant predictive biomarker for a better management of patients. Additionally, the study provides robust evidence for the regulatory role of the TGFβ/circLTBP2/miR-338-3p axis in iCCA. These findings provide a basis for further
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