Patent Application
20150267193
The compositions and methods described herein relate to microRNAs (miRs) and their role in various cancers. More particularly, compositions and methods described herein relate to miRs and their role in cancers influenced by the Wnt pathway and modulation thereof.
The citation of references herein shall not be construed as an admission that such is prior art to the present invention.
Wnts/wingless (wg) are a family of conserved signaling molecules that have been shown to regulate a plethora of fundamental developmental and cell biological processes, including cell proliferation, differentiation and cell polarity [Miller et al. Oncogene 18, 7860-72 (1999); Polakis. Genes Dev 14, 1837-51 (2000); Wodarz et al. Annu Rev Cell Dev Biol 14, 59-88 (1998)]. Mutations in the Wnt genes or in those genes encoding regulators of the Wnt/wg signaling pathway can cause devastating birth defects, including debilitating abnormalities of the central nervous system, axial skeleton, limbs, and occasionally other organs [Ciruna et al. Nature 439, 220-4 (2006); Grove et al. Development 125, 2315-25 (1998); Jiang et al. Dev Dyn 235, 1152-66 (2006); Kokubu et al. Development 131, 5469-80 (2004); Miyoshi et al. Breast Cancer Res 5, 63-8 (2003); Shu et al. Development 129, 4831-42 (2002); Staal et al. Hematol J 1, 3-6 (2000)]. Aberrant Wnt signaling has also been linked to human disease, such as hepatic, colorectal, breast and skin cancers [Miyoshi et al. supra (2003); Miyoshi et al. Oncogene 21, 5548-56 (2002); Moon et al. Nat Rev Genet 5, 691-701 (2004)]. Activating mutations of beta-catenin have also been found in around 5% of prostate cancers [Chesire et al., The Prostate 45, 323 (2000); Voeller et al., Cancer research 58, 2520 (1998)]. Mutation of APC has been found in 14% in one study [Gerstein et al., Genes, chromosomes & cancer 34, 9 (2002)] and 3% in another [Watanabe et al., Japanese journal of clinical oncology 26, 77 (1996)]. Over 20% of advanced prostate cancer, 77% of prostatic lymph node metastases and 85% of prostatic skeletal metastases have been reported to exhibit increased nuclear beta-catenin, as shown by immunohistochemistry [Chen et al., Cancer 101, 1345 (2004)]. The ligands of Wnt-pathway, Wnt1, Wnt2 and Wnt5a are, moreover, up-regulated in prostate cancer samples [Chen et al., Cancer 101, 1345 (2004); Katoh, International journal of oncology 19, 1003 (2001); Usui et al., Nihon Sanka Fujinka Gakkai zasshi 44, 703 (1992)]. Immunohistochemistry has revealed that one inhibitor of the Wnt-pathway, WIF1, was down-regulated in prostate cancer [Wissmann et al., The Journal of pathology 201, 204 (2003)].
Colon and gastrointestinal cancers are amongst the leading causes of cancer-related mortality and they all have been linked, together with many other cancers, to mutations in components of the Wnt/β-catenin pathway [1]. Therefore there is a major interest in targeting the activity of this pathway using genetic and chemical therapeutic tools. The promise of one emerging approach rests upon the therapeutic potential of small interfering RNAs (siRNAs) and microRNAs (miRs). miRs are small RNAs (˜ca. 22 nt in length) that regulate the level of mRNAs and proteins by targeted degradation of specific mRNAs and/or repression of their translation [2], [3]. Functions of miRs have been identified in apoptosis, proliferation, differentiation [2] and stem cell maintenance [4]. They have also been associated with cancer progression and metastasis [5], [6], [7]. Steady-state expression profiles of certain miRs have often been found to be deregulated in cancers and can aid in prognosis [8], [9], [10]. Individual miRs that have been reported to down-regulate oncogenes such as ras [11] are called anti-oncomiRs and inhibit cancer proliferation. Others, termed oncomiRs, function in a cancer-supportive or inductive manner by down-regulating tumor-suppressors such as p53 [12], [13] and inducing proliferation and/or metastasis. The Wnt/β-catenin pathway is often found to be elevated in gastrointestinal, breast and colon cancers among others and there is strong evidence for a role of hyper-activated Wnt signaling in cancer initiation and progression[14], [15], [16], [17], [18]. The key element of Wnt signaling is the transcriptional co-activator role of β-catenin, whose level is tightly controlled by a destruction complex including a scaffold protein, Axin-1, APC, and GSK-3β, a kinase that phosphorylates β-catenin, which results in its ubiquitination and subsequent proteasomal degradation[18], [19]. Wnt signaling via LRP5/6/Frizzled receptors and cytosolic Dsh among other factors, destabilizes this destruction complex, which leads to accumulation of β-catenin and its association with TCF/LEF family transcription factors in the nucleus to activate specific target genes [18], [19]. Negative regulators of Wnt signaling like APC and Axin function as tumor-suppressors and the viability of some cancer cell lines is believed to be Wnt-dependent [20], [21], [22], [23], [24], [25].
Several publications and patent documents are referenced in this application in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications and documents is incorporated by reference herein.
MicroRNAs (miRs) and the Wnt pathway are known to be dysregulated in human cancers and play key roles during cancer initiation and progression. To identify miRs that can modulate the activity of the Wnt pathway we performed a cell-based overexpression screen of 470 miRs in human HEK293 cells. We identified 38 candidate miRs that either activate or repress the Wnt pathway. A literature survey of all verified candidate miRs revealed that the Wnt-repressing miRs tend to be anti-oncomiRs and down-regulated in cancers while Wnt-activating miRs tend to be oncomiRs and upregulated during tumorigenesis. Epistasis-based functional validation of three candidate miRs, miR-1, miR-25 and miR-613, confirmed their inhibitory role in repressing the Wnt pathway and suggest that while miR-25 may function at the level of β-catenin (β-cat), miR-1 and miR-613 act upstream of β-cat. Both miR-25 and miR-1 inhibit cell proliferation and viability during selection of human colon cancer cell lines that exhibit dysregulated Wnt signaling. Finally, transduction of miR-1 expressing lentiviruses into primary mammary organoids derived from Conductin-lacZ mice significantly reduced the expression of the Wnt-sensitive β-gal reporter. In summary, these findings suggest the potential use of Wnt-modulating miRs as diagnostic and therapeutic tools in Wnt-dependent diseases, such as cancer.
In accordance with these findings, a method for modulating Wnt signaling pathways in a cell is presented, the method comprising contacting the cell with at least one microRNA listed in
In a particular embodiment thereof, the cell is a cancer cell. In a more particular embodiment, the Wnt signaling pathway is elevated or hyper-activated in the cancer cell and the at least one microRNA reduces or inhibits Wnt signaling pathways. In a further embodiment the at least one microRNA is microRNA 223 (SEQ ID NO: 1), 139 (SEQ ID NO: 3), 25 (SEQ ID NO: 4), 136 (SEQ ID NO: 5), 375 (SEQ ID NO: 7), 613 (SEQ ID NO: 11), 134 (SEQ ID NO: 13), 23a (SEQ ID NO: 15), 221 (SEQ ID NO: 16), 28 (SEQ ID NO: 17), 617 (SEQ ID NO: 19), 422b (SEQ ID NO: 20), 150 (SEQ ID NO: 27), 126 (SEQ ID NO: 30), 1-2 (SEQ ID NO: 35), 335 (SEQ ID NO: 36), 218-1 (SEQ ID NO: 37), or 9 (SEQ ID NO: 38). In another embodiment, the at least one microRNA comprises a consensus sequence as set forth in
In a further embodiment, the method for modulating Wnt signaling pathways in a cell is performed by contacting with or expressing at least one microRNA that activates the Wnt signaling pathway. Applications for which increasing or activating the Wnt signaling pathway is desirable include the treatment of bone degenerative disorders wherein bones become progressively more brittle. Osteoporosis is an exemplary bone degenerative disorder treatable using the Wnt pathway activating microRNA described herein. In a further embodiment thereof, the at least one microRNA is microRNA 196a-1 (SEQ ID NO: 2), 576 (SEQ ID NO: 6), 346 (SEQ ID NO: 8), 504 (SEQ ID NO: 9), 511-2 (SEQ ID NO: 10), 493-3p (SEQ ID NO: 12), 380-5p (SEQ ID NO: 14), 382 (SEQ ID NO: 18), 519e (SEQ ID NO: 21), 519b (SEQ ID NO: 22), 517a (SEQ ID NO: 23), 302a (SEQ ID NO: 24), 302d (SEQ ID NO: 25), 371 (SEQ ID NO: 26), 19b-1 (SEQ ID NO: 28), 555 (SEQ ID NO: 29), 106a (SEQ ID NO: 33), or 512-2-3p (SEQ ID NO: 34). In a more particular embodiment, the at least one microRNA is microRNA 576 (SEQ ID NO: 6), 346 (SEQ ID NO: 8), 504 (SEQ ID NO: 9), 511-2 (SEQ ID NO: 10), 493-3p (SEQ ID NO: 12), 380-5p (SEQ ID NO: 14), 382 (SEQ ID NO: 18), 519e (SEQ ID NO: 21), 519b (SEQ ID NO: 22), 302d (SEQ ID NO: 25), 371 (SEQ ID NO: 26), 555 (SEQ ID NO: 29), or 512-2-3p (SEQ ID NO: 34). In another embodiment, the at least one microRNA comprises a consensus sequence as set forth in
In a further aspect, a method for treating a subject with a cancer associated with an elevated or hyper-activated Wnt signaling pathway is presented, the method comprising administering to the subject at least one inhibitory microRNA shown in either of
In a still further aspect, a method for reducing or inhibiting Wnt signaling pathways in a subject with a cancer associated with an elevated or hyper-activated Wnt signaling pathway is presented, the method comprising administering to the subject at least one inhibitory microRNA shown in either of
In an embodiment of these methods, the at least one inhibitory microRNA is microRNA 223 (SEQ ID NO: 1), 139 (SEQ ID NO: 3), 25 (SEQ ID NO: 4), 136 (SEQ ID NO: 5), 375 (SEQ ID NO: 7), 613 (SEQ ID NO: 11), 134 (SEQ ID NO: 13), 23a (SEQ ID NO: 15), 221 (SEQ ID NO: 16), 28 (SEQ ID NO: 17), 617 (SEQ ID NO: 19), 422b (SEQ ID NO: 20), 150 (SEQ ID NO: 27), 126 (SEQ ID NO: 30), 1-2 (SEQ ID NO: 35), 335 (SEQ ID NO: 36), 218-1 (SEQ ID NO: 37), or 9 (SEQ ID NO: 38). In another embodiment, the at least one microRNA comprises a consensus sequence as set forth in
In a further aspect, the at least one inhibitory microRNA utilized in the methods described herein is modified to improve therapeutic efficacy as described herein and known in the art. In a particular embodiment thereof, the 3′ or 5′ end is modified to improve therapeutic efficacy. Delivery of the at least one microRNA may be achieved using a variety of means, including liposomes, lipidoids, nanovesicles and the like as described herein and known in the art.
MicroRNA described herein, whether inhibitory microRNA or activating microRNA with respect to their effect on Wnt signaling pathways, can be administered by a variety of routes, including without limitation, intravenously, intraperitoneally, orally, and/or in a localized fashion so as to delivery the microRNA in a region of the body wherein it is desirable to modulate the activity of Wnt signaling pathways. With regard to a method for reducing or inhibiting Wnt signaling pathways in a subject with a cancer associated with an elevated or hyper-activated Wnt signaling pathway or for treating a cancer associated with an elevated or hyper-activated Wnt signaling pathway, it may be desirable to deliver an inhibitory microRNA intratumorally or in a localized fashion in the immediate area of the tumor.
Also encompassed herein is a method for diagnosing a cancer associated with an elevated or hyper-activated Wnt signaling pathway in a subject, the method comprising determining the level of at least one microRNA shown in either of
Also encompassed herein is a method for determining the efficacy of a therapeutic agent for treating a cancer associated with an elevated or hyper-activated Wnt signaling pathway in a subject, the method comprising determining the level of at least one microRNA shown in either of
Also encompassed herein is a method for determining the efficacy of a therapeutic agent for treating a cancer associated with an elevated or hyper-activated Wnt signaling pathway in a subject, the method comprising determining the level of at least one microRNA shown in either of
Methods described herein may be performed using a sample isolated from the subject. Samples include, without limitation, fresh frozen or fixed tissue (e.g., paraffin-fixed tissue) of tumor biopsies collected during surgery. In a particular embodiment, the subject is a mammal. In a more particular embodiment, the mammal is a human.
Also envisioned herein is a method for screening to identify a modulator of at least one microRNA shown in either of
In a further aspect, at least one inhibitory microRNA according to either of
In another aspect, use of at least one inhibitory microRNA according to either of
In embodiments thereof, the at least one inhibitory microRNA for use in treating a cancer associated with an elevated or hyper-activated Wnt signaling pathway in a subject or use of the at least one inhibitory microRNA in the preparation of a medicament for treating a cancer associated with an elevated or hyper-activated Wnt signaling pathway in a subject calls for an inhibitory microRNA wherein the microRNA is microRNA 223 (SEQ ID NO: 1), 139 (SEQ ID NO: 3), 25 (SEQ ID NO: 4), 136 (SEQ ID NO: 5), 375 (SEQ ID NO: 7), 613 (SEQ ID NO: 11), 134 (SEQ ID NO: 13), 23a (SEQ ID NO: 15), 221 (SEQ ID NO: 16), 28 (SEQ ID NO: 17), 617 (SEQ ID NO: 19), 422b (SEQ ID NO: 20), 150 (SEQ ID NO: 27), 126 (SEQ ID NO: 30), 1-2 (SEQ ID NO: 35), 335 (SEQ ID NO: 36), 218-1 (SEQ ID NO: 37), or 9 (SEQ ID NO: 38). In another embodiment, the at least one inhibitory microRNA comprises a consensus sequence as set forth in
In a further aspect, the at least one inhibitory microRNA for use in treating a cancer associated with an elevated or hyper-activated Wnt signaling pathway or for use in the preparation of a medicament for treating a cancer associated with an elevated or hyper-activated Wnt signaling pathway is modified to improve therapeutic efficacy as described herein and known in the art. In a particular embodiment thereof, the 3′ or 5′ end is modified to improve therapeutic efficacy. The at least one inhibitory microRNA may be encapsulated in a liposome, lipidoid, or nanovesicle delivery vehicle. The at least one inhibitory microRNA may be administered via intradermal, intramuscular, intravenous, intraperitoneal, intratumoral, oral, rectal, buccal, or intranasal administration and/or in a localized fashion in the immediate area of the tumor.
In a further aspect, at least one activator/enhancer microRNA according to either of
In a further aspect, the at least one activator/enhancer microRNA for use in treating a bone degenerative disorder or for use in the preparation of a medicament for treating a bone degenerative disorder is modified to improve therapeutic efficacy as described herein and known in the art. In a particular embodiment thereof, the 3′ or 5′ end is modified to improve therapeutic efficacy. The at least one activator/enhancer microRNA may be encapsulated in a liposome, lipidoid, or nanovesicle delivery vehicle. The at least one activator/enhancer microRNA may be administered via intradermal, intramuscular, intravenous, intraperitoneal, intratumoral, oral, rectal, buccal, or intranasal administration and/or in a localized fashion in the immediate area of the osteoporotic bone.
Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing detailed description, which proceeds with reference to the following illustrative drawings, and the attendant claims.
miRNAs are a class of endogenous, small non-protein coding RNA molecules that regulate gene expression on a post-transcriptional level. miRNAs play a role in a variety of biological functions, including cellular proliferation, differentiation and apoptosis. The mechanism of miRNA mediated regulation of gene targets largely depends on the degree of complementarity between the miRNA and its target or targets. Typically, miRNAs that bind to mRNA targets with imperfect complementarity bring about translational repression of gene targets, while miRNAs that bind to their mRNA targets with perfect complementarity induce target mRNA cleavage.
Emerging evidence suggests that miRNAs play important roles in the carcinogenesis of various human cancers. Indeed, some miRNAs may be involved in cancers as oncogenes and/or tumor suppressors, whereas others are implicated in tumor invasion and metastasis. See, for example, Xia (J Cancer Molecules 4:79-89, 2008) and Budhu et al. (J Hematology & Oncology 3:37, 2010), the entire contents of each of which is incorporated herein in its entirety.
Further to the above, it has been recently suggested that the delivery and use of anti-oncomiRs or inhibiting oncomiR functionality with antagomiRs [26] may serve as a promising therapeutic approach [27]. We therefore hypothesized that identifying and characterizing miRs that specifically modulate the Wnt pathway could provide a basis for the development of novel Wnt-based therapeutics in Wnt-associated diseases, such as cancer. Research in the past few years have implicated some miRs in the regulation of Wnt signaling [28], [29], [30], [31], [32], [33], [34]. Here we report a systematic screening of a library of 470 human synthetic Pre-miRs and identification of 38 miRs that modulate the activity of the Wnt pathway in human HEK293 cells. Secondary validation and functional testing of 3 candidate miRs, namely miR-1, miR-25 and miR-613 confirmed their inhibitory effect on the activity of the Wnt pathway. Epistasis experiments revealed that miR-1 and miR-613 target the pathway upstream of Axin or active β-catenin, and that miR-25 acts downstream, at the level of β-cat, likely by targeting β-cat's coding sequence. Importantly, overexpression of miR-25 and miR-1 inhibited proliferation/viability of human colon cancer cells that are known to be dependent on sustained β-cat signaling for their survival[22], [24]. Furthermore, expression of miR-1 in primary mammary epithelial organoids derived from a Wnt-reporter mouse (conductin-lacZ) significantly reduced the expression of the β-gal reporter. These results suggest that these candidate miRs may influence Wnt signaling activity in vivo.
Accordingly, candidate miRs described herein provide novel biomarkers for cancer diagnosis, classification, and prognostic evaluation. Candidate miRs are also envisioned herein as potential therapeutic agents, particularly with respect to disorders relating to the Wnt signaling pathway, such as cancer. Candidate miRs described herein may also be used in screening assays to identify modulators of their activity, which modulators can be used as therapeutic agents for treating disorders relating to aberrant activity of the Wnt signaling pathway.
In order to more clearly set forth the parameters of the present invention, the following definitions are used:
As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus for example, reference to “the method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure.
The term “complementary” refers to two DNA strands that exhibit substantial normal base pairing characteristics. Complementary DNA may, however, contain one or more mismatches. The term “hybridization” refers to the hydrogen bonding that occurs between two complementary DNA strands.
“Nucleic acid” or a “nucleic acid molecule” as used herein refers to any DNA or RNA molecule, either single or double stranded and, if single stranded, the molecule of its complementary sequence in either linear or circular form. In discussing nucleic acid molecules, a sequence or structure of a particular nucleic acid molecule may be described herein according to the normal convention of providing the sequence in the 5′ to 3′ direction. With reference to nucleic acids of the invention, the term “isolated nucleic acid” is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous in the naturally occurring genome of the organism in which it originated. For example, an “isolated nucleic acid” may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism.
When applied to RNA, the term “isolated nucleic acid” refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it is generally associated in its natural state (i.e., in cells or tissues). An isolated nucleic acid (either DNA or RNA) may further represent a molecule produced directly by biological or synthetic means and separated from other components present during its production.
The terms “percent similarity”, “percent identity” and “percent homology” when referring to a particular sequence are used as set forth in the University of Wisconsin GCG software program and are known in the art.
The term “functional” as used herein implies that the nucleic or amino acid sequence is functional for the recited assay or purpose.
The term “functional fragment” as used herein implies that the nucleic or amino acid sequence is a portion or subdomain of a full length polypeptide and is functional for the recited assay or purpose.
The phrase “consisting essentially of” when referring to a particular nucleotide or amino acid means a sequence having the properties of a given SEQ ID NO. For example, when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the basic and novel characteristics of the sequence.
A “replicon” is any genetic element, for example, a plasmid, cosmid, bacmid, phage or virus, that is capable of replication largely under its own control. A replicon may be either RNA or DNA and may be single or double stranded.
A “vector” is a replicon, such as a plasmid, cosmid, bacmid, phage or virus, to which another genetic sequence or element (either DNA or RNA) may be attached so as to bring about the replication of the attached sequence or element.
An “expression vector” or “expression operon” refers to a nucleic acid segment that may possess transcriptional and translational control sequences, such as promoters, enhancers, translational start signals (e.g., ATG or AUG codons), polyadenylation signals, terminators, and the like, and which facilitate the expression of a polypeptide coding sequence in a host cell or organism.
A DNA “coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3′ to the coding sequence.
A “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease Si), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the −10 and −35 consensus sequences.
An “expression control sequence” is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is “under the control” of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.
A “signal sequence” can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.
As used herein, the term “operably linked” refers to a regulatory sequence capable of mediating the expression of a coding sequence and which is placed in a DNA molecule (e.g., an expression vector) in an appropriate position relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements (e.g. promoters, enhancers, and termination elements) in an expression vector. This definition is also sometimes applied to the arrangement of nucleic acid sequences of a first and a second nucleic acid molecule wherein a hybrid nucleic acid molecule is generated.
The term “oligonucleotide,” as used herein refers to primers and probes of the present invention, and is defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably more than three. The exact size of the oligonucleotide will depend on various factors and on the particular application and use of the oligonucleotide.
The term “probe” as used herein refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe. A probe may be either single-stranded or double-stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide probe typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides. The probes herein are selected to be “substantially” complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to “specifically hybridize” or anneal with their respective target strands under a set of pre-determined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non-complementary nucleotide fragment may be attached to the 5′ or 3′ end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically.
The term “specifically hybridize” refers to the association between two single-stranded nucleic acid molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed “substantially complementary”). In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.
The term “primer” as used herein refers to an oligonucleotide, either RNA or DNA, either single-stranded or double-stranded, either derived from a biological system, generated by restriction enzyme digestion, or produced synthetically which, when placed in the proper environment, is able to functionally act as an initiator of template-dependent nucleic acid synthesis. When presented with an appropriate nucleic acid template, suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as a suitable temperature and pH, the primer may be extended at its 3′ terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield a primer extension product. The primer may vary in length depending on the particular conditions and requirement of the application. For example, in diagnostic applications, the oligonucleotide primer is typically 15-25 or more nucleotides in length. The primer must be of sufficient complementarity to the desired template to prime the synthesis of the desired extension product, that is, to be able anneal with the desired template strand in a manner sufficient to provide the 3′ hydroxyl moiety of the primer in appropriate juxtaposition for use in the initiation of synthesis by a polymerase or similar enzyme. It is not required that the primer sequence represent an exact complement of the desired template. For example, a non-complementary nucleotide sequence may be attached to the 5′ end of an otherwise complementary primer. Alternatively, non-complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to functionally provide a template-primer complex for the synthesis of the extension product.
Primers may be labeled fluorescently with 6-carboxyfluorescein (6-FAM). Alternatively primers may be labeled with 4, 7, 2′,7′-Tetrachloro-6-carboxyfluorescein (TET). Other alternative DNA labeling methods are known in the art and are contemplated to be within the scope of the invention.
The term “substantially pure” refers to a preparation comprising at least 50-60% by weight of a given material (e.g., nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-95% by weight of the given compound. Purity is measured by methods appropriate for the given compound (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like). “Mature protein” or “mature polypeptide” shall mean a polypeptide possessing the sequence of the polypeptide after any processing events that normally occur to the polypeptide during the course of its genesis, such as proteolytic processing from a polypeptide precursor. In designating the sequence or boundaries of a mature protein, the first amino acid of the mature protein sequence is designated as amino acid residue 1.
The term “tag”, “tag sequence” or “protein tag” refers to a chemical moiety, either a nucleotide, oligonucleotide, polynucleotide or an amino acid, peptide or protein or other chemical, that when added to another sequence, provides additional utility or confers useful properties to the sequence, particularly with regard to methods relating to the detection or isolation of the sequence. Thus, for example, a homopolymer nucleic acid sequence or a nucleic acid sequence complementary to a capture oligonucleotide may be added to a primer or probe sequence to facilitate the subsequent isolation of an extension product or hybridized product. In the case of protein tags, histidine residues (e.g., 4 to 8 consecutive histidine residues) may be added to either the amino- or carboxy-terminus of a protein to facilitate protein isolation by chelating metal chromatography. Alternatively, amino acid sequences, peptides, proteins or fusion partners representing epitopes or binding determinants reactive with specific antibody molecules or other molecules (e.g., flag epitope, c-myc epitope, transmembrane epitope of the influenza A virus hemaglutinin protein, protein A, cellulose binding domain, calmodulin binding protein, maltose binding protein, chitin binding domain, glutathione S-transferase, and the like) may be added to proteins to facilitate protein isolation by procedures such as affinity or immunoaffinity chromatography. Chemical tag moieties include such molecules as biotin, which may be added to either nucleic acids or proteins and facilitates isolation or detection by interaction with avidin reagents, and the like. Numerous other tag moieties are known to, and can be envisioned by, the trained artisan, and are contemplated to be within the scope of this definition.
The terms “transform”, “transfect”, “transduce”, shall refer to any method or means by which a nucleic acid is introduced into a cell or host organism and may be used interchangeably to convey the same meaning. Such methods include, but are not limited to, transfection, electroporation, microinjection, PEG-fusion and the like.
The introduced nucleic acid may or may not be integrated (covalently linked) into nucleic acid of the recipient cell or organism. In bacterial, yeast, plant and mammalian cells, for example, the introduced nucleic acid may be maintained as an episomal element or independent replicon such as a plasmid. Alternatively, the introduced nucleic acid may become integrated into the nucleic acid of the recipient cell or organism and be stably maintained in that cell or organism and further passed on or inherited to progeny cells or organisms of the recipient cell or organism. In other applications, the introduced nucleic acid may exist in the recipient cell or host organism only transiently.
The compositions containing the molecules or compounds of the invention can be administered for diagnostic and/or therapeutic treatments. In therapeutic applications, compositions are administered to a patient already suffering from a hyperproliferative disorder (such as, e.g., cancer) in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. An amount adequate to accomplish this is defined as a “therapeutically effective amount or dose.” Amounts effective for this use will depend on the severity of the disease and the weight and general state of the patient.
As used herein, the term “cancer” refers to an abnormal growth of tissue resulting from uncontrolled progressive multiplication of cells. Examples of cancers that can be treated according to a method of the present invention include cancers associated with altered Wnt/wg pathway signaling. Such conditions include a variety of hyperproliferative disorders and cancers, including prostate cancer, breast cancer, skin cancer (e.g., melanoma), colorectal cancer, hepatic cancer (e.g., hepatocellular cancer and hepatoblastoma), head and neck cancer, lung cancer (e.g., non-small cell lung cancer), gastric cancer, mesothelioma, synovial sarcoma, cervical cancer, endometrial ovarian cancer, Wilm's tumor, bladder cancer and leukemia. In a particular embodiment, cancers that can be treated according to a method of the present invention include prostate cancer, breast cancer, skin cancer (e.g., melanoma), and colorectal cancer. In a more particular embodiment, cancers that can be treated according to a method of the present invention include prostate cancer, breast cancer, and skin cancer (e.g., melanoma). See, for example, Luu et al. (2004, Current Cancer Drug Targets 4:653), Lepourcelet et al. (2004, Cancer Cell 5:91), Barker and Clevers (2006, Nature Reviews Drug Discovery 5:997), and Watanabe and Dai (2011, Proc Natl Acad Sci 108:5929), the entire content of each of which is incorporated herein by reference.
As used herein, an “agent”, “candidate compound”, or “test compound” may be used to refer to, for example, nucleic acids (e.g., DNA and RNA), carbohydrates, lipids, proteins, peptides, peptidomimetics, small molecules and other drugs.
The term “control substance”, “control agent”, or “control compound” as used herein refers a molecule that is inert or has no activity relating to an ability to modulate a biological activity. With respect to the present disclosure, such control substances are inert with respect to an ability to modulate a Wnt signaling pathway. Exemplary controls include, but are not limited to, solutions comprising physiological salt concentrations.
The term ‘treating’ or ‘treatment’ of any disease, condition or infection refers, in one embodiment, to ameliorating the disease or infection (i.e., arresting the disease or growth of the infectious agent or bacteria or reducing the manifestation, extent or severity of at least one of the clinical symptoms thereof). In another embodiment ‘treating’ or ‘treatment’ refers to ameliorating at least one physical parameter, which may not be discernible by the subject. In yet another embodiment, ‘treating’ or ‘treatment’ refers to modulating the disease or infection, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In a further embodiment, ‘treating’ or ‘treatment’ relates to slowing the progression of a disease.
The subject is preferably an animal, including but not limited to animals such as mice, rats, cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, more preferably a primate, and most preferably a human.
The phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
Methods and agents for modulating Wnt signaling pathways in a cell are presented herein. Such agents include the microRNA species listed in
Exemplary microRNAs identified herein as inhibitors of Wnt signaling pathways and listed in
Exemplary microRNAs identified herein as inhibitors of Wnt signaling pathways include members of the miR-1/206 family and the miR-25/92 family. See, for example,
Exemplary microRNAs identified herein as activators/enhancers of Wnt signaling pathways include microRNA 196a-1 (SEQ ID NO: 2), 576 (SEQ ID NO: 6), 346 (SEQ ID NO: 8), 504 (SEQ ID NO: 9), 511-2 (SEQ ID NO: 10), 493-3p (SEQ ID NO: 12), 380-5p (SEQ ID NO: 14), 382 (SEQ ID NO: 18), 519e (SEQ ID NO: 21), 519b (SEQ ID NO: 22), 517a (SEQ ID NO: 23), 302a (SEQ ID NO: 24), 302d (SEQ ID NO: 25), 371 (SEQ ID NO: 26), 19b-1 (SEQ ID NO: 28), 555 (SEQ ID NO: 29), 106a (SEQ ID NO: 33), and 512-2-3p (SEQ ID NO: 34). In a more particular embodiment, the activator/enhancer microRNA is 576 (SEQ ID NO: 6), 346 (SEQ ID NO: 8), 504 (SEQ ID NO: 9), 511-2 (SEQ ID NO: 10), 493-3p (SEQ ID NO: 12), 380-5p (SEQ ID NO: 14), 382 (SEQ ID NO: 18), 519e (SEQ ID NO: 21), 519b (SEQ ID NO: 22), 302d (SEQ ID NO: 25), 371 (SEQ ID NO: 26), 555 (SEQ ID NO: 29), or 512-2-3p (SEQ ID NO: 34). In a further embodiment thereof, the activator/enhancer microRNA is a member of the miR-302 family and the miR-515 family. A consensus sequence shared in common among these families is GUGCNUCCN(N)(N)UUU(N)NNGN. See, for example,
Methods and agents for treating a subject with a cancer associated with an elevated or hyper-activated Wnt signaling pathway are also presented herein. MicroRNAs identified as inhibitors of Wnt signaling pathways have application for such purposes. As indicated herein, microRNAs identified herein as inhibitors of Wnt signaling pathways include members of the miR-1/206 family and the miR-25/92 family. See, for example,
Methods and agents for treating a subject with a bone degenerative disorder (such as osteoporosis) are also presented herein. MicroRNAs identified as activators/enhancers of Wnt signaling pathways have application for such purposes. As indicated herein, microRNAs identified herein as activators/enhancers of Wnt signaling pathways include members of the miR-302 family and the miR-515 family. A consensus sequence shared in common among these families is GUGCNUCCN(N)(N)UUU(N)NNGN. See, for example,
Methods for expressing microRNAs described herein and expression vectors encoding same are described herein in the Examples section and are, furthermore, known in the art. Exemplary expression vectors include, without limitation, pcDNA3.1(−) vector; retroviral RNAi vectors, including lentiviral vectors such as, e.g., pLV and pCDH-CMV-MSC-EF1-Puro lentiviral vector [See, for example, Yang et al. (Oncogene 22:5694, 2003); Zhou et al. (Oncogene 31:2968-2978, 2012); the entire content of each of which is incorporated herein by reference) and oncoretrovirus vectors; adeno-associated virus (AAV) vectors, particularly those that include a self-complementary genome that enhances therapeutic gene expression and non-human primate AAV serotypes that facilitate efficient transduction following vascular delivery (See, for example, Kota et al. Cell 137:1005, 2009; the entire content of which is incorporated herein by reference); recombinant adenoviral vectors (See, for example, Xia et al. (Nature Biotechnology 20:106, 2002; the entire content of which is incorporated herein by reference); polycistronic RNA polymerase II expression vectors such as those described by Chung et al. (Nucleic Acids Res 34:e53, 2006, the entire content of which is incorporated herein by reference); and tet inducible vector systems. See also Devroe et al. (Expert Opin in Biol Therapy 4:319-327, 2004; the entire content of which is incorporated herein by reference along with references cited therein) for a review expression vectors suitable for delivery of RNAi therapeutics.
Liposomes composed of the neutral lipid 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) have, moreover, been used successfully to deliver an EphA2-targeting siRNA in an orthotopic mouse model of ovarian cancer. Therapeutic delivery of the EphA2-targeting siRNA resulted in decreased EphA2 expression in the tumor and decreased tumor growth when combined with chemotherapy. See, for example, Landen et al. (Cancer Res. 65:6910, 2005); Merritt et al. (J Natl Cancer Inst 100:359, 2008), the entire content of each of which is incorporated herein by reference). In view of the similarities between siRNA and miRNA, it is reasonable to use liposomes composed of DOPC for delivery of the miRNA species desribed herein.
Lipidoids such as those described by Akinc et al. (Nature Biotechnology 26:561-569, 2008; the entire content of which is incorporated herein) for delivery of RNAi therapeutics are also envisioned herein. Akinc et al. demonstrated that the lipidoids described there have broad utility for both local and systemic delivery of RNA therapeutic, both siRNA and miRNA. The safety and efficacy of lipidoids were, moreover, evaluated and confirmed in three animal models: mice, rats and nonhuman primates. Compositions and methods useful for administering nucleic acid based therapies are also described in U.S. Pat. No. 8,034,376, the entire content of which is incorporated herein in its entirety.
Cholesterol conjugation of siRNA has also been demonstrated to improve significantly in vivo pharmacological properties of siRNA. See, for example, Soutschek et al. (Nature 432:173, 2004), the entire content of which is incorporated herein by reference.
Delivery of microRNA may be enhanced by packaging in a nanovesicle or other vehicle developed for microRNA delivery to target tissue in the subject. Nanoparticles have been used successfully for targeted delivery in vitro and in vivo and have, moreover, exhibited reduced toxicity when compared to other therapeutic delivery vehicles. Nanoparticles have been developed for administration via injection and oral consumption. Bisht et al. (Mol Cancer Ther 7:3878, 2008), for example, describe the synthesis and physicochemical characterization of orally bioavailable polymeric nanoparticles composed of N-isopropylacrylamide, methylmethacrylate, and acrylic acid in the molar ratios of 60:20:20 (designated NMA622). Amphiphilic NMA622 nanoparticles show a size distribution of <100 nm (mean diameter of 80+/−34 nm) with low polydispersity and can readily encapsulate a number of poorly water-soluble drugs, including drugs such as rapamycin that are used for treating various cancers, within the hydrophobic core. Mice receiving as much as 500 mg/kg of the orally administered void NMA622 for 4 weeks did not exhibit detectable systemic toxicity. NMA622 nanoparticles, therefore, provide a suitable platform for oral delivery of water-insoluble drugs for cancer therapy and thus, may find application in the delivery of miRNA.
Delivery of RNA therapeutics has also been mediated by direct conjugation of delivery agents to the RNA moiety, formulation using lipid polymer or peptide-based delivery systems and the formation of complexes with antibody fusion proteins. See, for example, Akinc et al. (Nature Biotechnology 26:561-569, 2008) the entire content of which, including references cited therein, is incorporated herein.
Agents described herein, such as, for example, a microRNA may be labelled with a detectable or functional label. Detectable labels include, but are not limited to, radiolabels such as the isotopes 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 121I, 124I, 125I, 131I, 111In, 117Lu, 211At, 198Au, 67CU, 225Ac, 213Bi, 99Tc and 186Re, which may be attached to agents using conventional chemistry known in the art. Labels also include fluorescent labels (for example fluorescein, rhodamine, Texas Red) and labels used conventionally in the art for MRI-CT imaging. They also include enzyme labels such as horseradish peroxidase, β-glucoronidase, β-galactosidase, and urease. Labels further include chemical moieties such as biotin which may be detected via binding to a specific cognate detectable moiety, e.g. labelled avidin. Functional labels include substances which are designed to be targeted to the site of a tumor to facilitate targeted delivery of a microRNA thereto or cause destruction of tumor tissue. Such functional labels include cytotoxic drugs such as 5-fluorouracil or ricin and enzymes such as bacterial carboxypeptidase or nitroreductase, which are capable of converting prodrugs into active drugs at the site of a tumor.
Methods for diagnosing a cancer associated with an elevated or hyper-activated Wnt signaling pathway in a subject are also encompassed herein, wherein such methods comprise determining the level of at least one microRNA shown in either of
Methods for determining the efficacy of a therapeutic agent for treating a cancer associated with an elevated or hyper-activated Wnt signaling pathway in a subject are also described, the method comprising determining the level of at least one microRNA shown in either of
With regard to methods directed to diagnosis, a sample may be isolated from the subject and analyzed to assess/detect levels of the at least one microRNA. Methods directed to determining the efficacy of a therapeutic agent for treating a cancer associated with an elevated or hyper-activated Wnt signaling pathway in a subject, for example, may be performed using a sample isolated from the subject. Such samples would include fresh frozen or even fixed tissues collected during surgery. Because miRNA are very stable and therefore resistant to degradation, they can be successfully isolated from tumor biopsies and paraffin-fixed tissues.
Identification of particular microRNA that are causally linked with cancers associated with aberrant Wnt signaling will provide information with which a clinician can tailor therapeutic intervention for a patient afflicted with the cancer. If, for example, a patient has a Wnt related cancer wherein an activator/enhancer microRNA described herein or plurality of same is upregulated (expressed at high levels relative to control), then therapeutic intervention can be particularly targeted to decrease levels of this microRNA/s. If, on the other hand, a patient has a Wnt related cancer wherein an inhibitor microRNA described herein or plurality of same is downregulated (expressed at low levels relative to control), then therapeutic intervention can be particularly targeted to increase levels of this microRNA/s. In cases, wherein a patient with a Wnt related cancer exhibits both increased levels of activator/enhancer microRNA described herein and decreased levels of inhibitor microRNA described herein, a skilled practitioner can tailor therapeutic intervention to both decrease levels of the activator/enhancer microRNA and increase levels of the inhibitor microRNA.
The microRNA species described herein may also be used in screening methods to identify a modulators of their activity of at least one microRNA shown in either of
The present agents are used as therapeutic agents for the treatment of conditions in mammals that are causally related or attributable to Wnt signaling pathways. Accordingly, the compounds and pharmaceutical compositions of this invention find use as therapeutics for modulating and/or treating a variety of conditions causally related or attributable to Wnt signaling pathways in mammals, including humans. In a particular aspect, microRNAs identified as inhibitors of Wnt signaling pathways are used as therapeutics for treating a cancer associated with an elevated or hyper-activated Wnt signaling pathway. Such cancers include, for example, breast cancer, prostate cancer, colorectal cancer, hepatic cancer, or skin cancer. In an another embodiment, microRNAs identified as activators/enahncers of Wnt signaling pathways are used as therapeutics for treating bone degenerative disorders, such as osteoporosis.
Agents described herein (e.g., micoRNA) may be administered to a patient in need of treatment via any suitable route, including, e.g., intradermal, intramuscular, intravenous, intraperitoneal, intratumoral, oral, rectal, buccal or intranasal administration. The precise dose will depend upon a number of factors, including whether the agents are for diagnosis or for treatment or for prevention. The dosage or dosing regimen of an adult patient may be proportionally adjusted for children and infants, and also adjusted for other administration or other formats, in proportion for example to molecular weight or immune response. Administration or treatments may be repeated at appropriate intervals, at the discretion of the physician.
Agents described herein are generally administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the agents. Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. intravenous, or by deposition at a tumor site.
Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
For intravenous injection, e.g., or injection at the site of affliction, the active ingredient may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included, as required.
A composition may be administered alone or in combination with other treatments, therapeutics or agents, either simultaneously or sequentially dependent upon the condition to be treated. In addition, the present invention contemplates and includes compositions comprising the agents herein described and other agents or therapeutics such as immune modulators, antibodies, immune cell stimulators, or adjuvants. In addition, the composition may be administered with hormones, such as dexamethasone, immune modulators, such as interleukins, tumor necrosis factor (TNF) or other growth factors, colony stimulating factors, or cytokines which stimulate the immune response and elimination of cancer cells. The composition may also be administered with, or may include combinations along with pathway specific cancer drugs, chemotherapeutics, radiation, and other agents used to treat cancers and known to those skilled in the art.
The preparation of therapeutic compositions which contain agents as described herein and/or polypeptides or analogs as active ingredients is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions. However, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified. The active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient.
Polypeptides for inclusion in compositions can be formulated into a therapeutic composition as neutralized pharmaceutically acceptable salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule), which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
Other materials as well as processing techniques and the like are set forth in Part 8 of Remington's Pharmaceutical Sciences, 17th edition, 1985, Mack Publishing Company, Easton, Pa., which is incorporated herein by reference.
Accordingly, also encompassed herein is a composition comprising at least one of the miRNA identified herein as an inhibitor of Wnt signaling pathways as listed in
Also encompassed herein is a composition comprising at least one of the miRNA identified herein as an activator/enhancer of Wnt signaling pathways as listed in
The agent containing compositions are conventionally administered intramuscularly, intravenously, as by injection of a unit dose, or orally, for example. The term “unit dose” when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.
The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated and the stage and type of cancer being treated. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. Suitable regimens for initial administration and follow on administration are also variable, and may include an initial administration followed by repeated doses at appropriate intervals by a subsequent injection or other administration.
The present invention further provides an isolated nucleic acid encoding an agent of the present invention. Nucleic acid includes DNA and RNA.
In a particular aspect, miRNA described herein is synthesized in accordance with standard procedures. miRNA can also be generated via expression constructs in cells, wherein the cell processes the transcript to generate the mature miRNA. Such methods are described in the Examples presented herein and known in the art.
Also envisioned for use in the methods and uses described herein are miRNA mimics, which are small, chemically modified double-stranded RNAs that mimic endogenous miRNAs. In accordance with the methods and uses described herein, an miRNA mimic of an miRNA species identified as an inhibitor of Wnt signaling pathways may, for example, be used to reduce or inhibit Wnt signaling pathways and/or treat a cancer associated with an elevated or hyper-activated Wnt signaling pathway.
In a further application, miRNA inhibitors are envisioned for use in the methods and uses described herein. miRNA inhibitors are small, chemically modified single-stranded RNA molecules designed to specifically bind to and inhibit endogenous miRNA molecules. In accordance with the methods and uses described herein, miRNA inhibitors may, for example, be used to down-regulate the activity of an miRNA species identified as an enhancer of Wnt signaling pathways and thus, reduce or inhibit Wnt signaling pathways and/or treat a cancer associated with an elevated or hyper-activated Wnt signaling pathway.
An exemplary inhibitory molecule of miRNA is an anti-miRNA oligonucleotide (AMO) which blocks the interactions between an miRNA and its target mRNAs by competition. AMOs may be chemically modified in a variety of ways to improve the stability. Locked nucleic acid (LNA), often referred to as inaccessible RNAs, refers to bicyclic high-affinity RNA analogs wherein the ribose moiety is chemically locked in a RNA-mimicking N-type (C3′-endo) conformation by the introduction of an extra 2′-O, 4′-C methylene bridge. The locked ribose conformation enhances base stacking and backbone preorganization and significantly increases the thermal stability of complexes upon hybridization with complementary single-stranded RNA target molecules. In addition, LNA is compatible with RNase H cleavage and displays high aqueous solubility and low toxicity in vivo. The 2′-O-methyl (2′-O-Me) modification, as well as the 2′-O-methoxyethyl (2′-MOE) and 2′-fluoro (2′-F) chemistries are modified at the 2′ position of the sugar moiety and oligonucleotides comprising these modifications have shown promise in functional inhibition of miRNAs. Nuclease resistance is also improved by backbone modification of the parent phosphodiester linkages into phosphorothioate (PS) linkages in which a sulfur atom replaces one of the non-bridging oxygen atoms in the phosphate group or by using morpholino oligomers, in which a six-membered morpholine ring replaces the sugar moiety In addition to chemical modifications, some improvement in inhibitor potency is observed by increasing the length of the AMOs. miRNA sponges, miRNA masking, and small molecule inhibitors are also envisioned herein. See, for example, Li et al. (AAPS J 11:747, 2009; the entire content of which is incorporated herein by reference along with references cited therein) for additional details.
Chemically modified synthetic miRNA mimics and inhibitors are available and commercially available from a variety of vendors. Such vendors include Ambion®, Qiagen, Life Technologies, GenePharma Biotech, and Thermo Fisher Scientific Dharmacon. Thermo Fisher Scientific Dharmacon, for example, can be used as a source for every human, mouse, and rat miRNA present in the miRBase Sequence Database to date (See worldwide web microrna.sanger.ac.uk). Qiagen, for example, can be used as a source of miScript miRNA Mimics, which are chemically synthesized, double-stranded RNAs that mimic mature endogenous miRNAs after transfection into cells. miScript miRNA Mimics are available at cell-culture grade (>90% purity) or animal grade (HPLC purified; for in vivo applications).
In another particular aspect, the present invention provides a nucleic acid which codes for or corresponds a miRNA of the invention as defined above, including any one of those listed in
The present invention also provides constructs in the form of plasmids, vectors, and transcription or expression cassettes which comprise at least one polynucleotide as above. The present invention also provides a recombinant host cell which comprises one or more constructs as above. A nucleic acid encoding any agent as provided herein forms an aspect of the present invention, as does a method of production of the agent which method comprises expression from encoding nucleic acid therefor. Expression may conveniently be achieved by culturing recombinant host cells containing the nucleic acid under appropriate conditions. Following production by expression, an agent may be isolated and/or purified using any suitable technique, then used as appropriate.
Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous miRNA or polypeptides include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, HEK293 cells, HCT116, and many others.
Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Short Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992. The disclosures of Sambrook et al. and Ausubel et al. are incorporated herein by reference.
Thus, a further aspect of the present invention provides a host cell containing nucleic acid as disclosed herein. A still further aspect provides a method comprising introducing such nucleic acid into a host cell. The introduction may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage. The introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene. The present invention also provides a method which comprises using a construct as stated above in an expression system in order to express a miRNA as described above.
Another feature of this invention is the expression of DNA sequences contemplated herein, particularly encoding the agents described herein. As is well known in the art, DNA sequences may be expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate unicellular host. A wide variety of host/expression vector combinations may be employed in expressing DNA sequences. Useful expression vectors, for example, may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids col El, pCR1, pBR322, pMB9 and their derivatives, plasmids such as RP4; phage DNAs, e.g., the numerous derivatives of phage λ, e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2μ plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.
Any of a wide variety of expression control sequences (sequences that control the expression of a DNA sequence operatively linked to it) may be used in these vectors to express DNA sequences. Such useful expression control sequences include, for example, the early or late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the LTR system, the major operator and promoter regions of phage λ, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), the promoters of the yeast-mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
A wide variety of unicellular host cells are also useful in expressing the DNA sequences of this invention. These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO, YB/20, NSO, SP2/0, R1.1, B-W and L-M cells, African Green Monkey kidney cells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g., Sf9), and human cells and plant cells in tissue culture.
It will be understood that not all vectors, expression control sequences and hosts will function equally well to express the DNA sequences of this invention. Neither will all hosts function equally well with the same expression system. However, one skilled in the art will be able to select the proper vectors, expression control sequences, and hosts without undue experimentation to accomplish the desired expression without departing from the scope of the invention. In selecting an expression control sequence, a variety of factors will normally be considered. These include, for example, the relative strength of the system, its controllability, and its compatibility with the particular DNA sequence or gene to be expressed, particularly as regards potential secondary structures. Suitable unicellular hosts will be selected by consideration of, e.g., their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly, and their fermentation requirements, as well as the toxicity to the host of the product encoded by the DNA sequences to be expressed, and the ease of purification of the expression products. Considering these and other factors a person skilled in the art will be able to construct a variety of vector/expression control sequence/host combinations that will express the DNA sequences on fermentation or in large scale animal culture.
The invention may be better understood by reference to the following non-limiting Examples, which are provided as exemplary of the invention. The following examples are presented in order to more fully illustrate the preferred embodiments of the invention and should in no way be construed, however, as limiting the broad scope of the invention.
Screening and Reporter Assays—
Screening and reporter assays were carried out as described previously [35]. Briefly, HEK293 cells trypsinized and resuspended in antibiotic-free culture media were plated and transfected in 384 well plates (Corning, Cat No. 3704). Transfections were conducted with 0.1 μL Lipofectamine 2000 (Invitrogen) and 27 ng STF19 reporter plasmid (a kind Gift from the R.T. Moon laboratory, Seattle, USA) together with 5 ng Renilla-CMV vector as internal control each well. 7 μL DNA-containing transfection-mix with serum-free DMEM and Lipofectamine2000 was added to the pre-plated Hsa-pre-miR™ in a 384 well plate (5 μL each well, 1.5 pmol, human pre-miR library, n=470, Ambion, #4385830) for a final concentration of 42.9 nM ca. 5000 viable HEK293 cells were plated in 23 μL culture media to the pre-dispensed transfection mixes and incubated for two days. 50 μL of pretested Wnt3a-conditioned media (harvested from L-Wnt3A cells [36], a kind gift from the laboratory or Dr. R. T. Moon, University of Washington, Seattle) were added at day 2 post-transfection and cells were incubated for additional 16 hours. Pre-miRs™ used are synthetic siRNA-like and modified strand selective small dsRNAs with verified specificity (Ambion). Cells were lysed in 20 μL DualGlo (Promega, #E2920) substrate buffer and normalized luminosity, which is the ratio of the firefly reporter (STF16x-Firefly) and the renilla luciferase (CMV-driven), was read with an EnVision multilabel plate-reader (Perkin Elmer). The ratio of the STF-firefly and CMV-renilla RLU (relative light units) for each Pre-miR was divided by the plate average (PA) or control siRNAs, respectively. The primary screen was performed in quadruplets for statistical/assay robustness. Cutoff values were based on the performance of control siRNAs with the only exception of the high/medium-scorer miR-200a that was also included into the cherrypick-listing because of its described role[29], [32]. Raw data, normalized values, and cherry-picking listings are deposited available at the NYU-RNAi core facility and available upon request. Other reporter assays, including epistasis experiments, were conducted in 96- or 384-well plates using the same protocol settings. STF19/CMV-RL activity in cancer cell lines was measured one day post-transfection.
Cloning, DNA and RNA Reagents
The coding region of human β-catenin (S37A mutant; transcript nt position 307-1874 NcoI-Klenow; NotI) was subcloned into the 3′UTR of the Renilla gene (NotI, SpeI-Klenow) within a psi-check-2 reporter vector with modified MCS to monitor the influence of miR-25 on its transcript stability and translation. In this assay a CMV-driven synthetic-firefly-luciferase served as internal control. A β-catenin fragment lacking exon-3 (543-1874) behaved similar (not shown). A human Pri-miR-25 fragment was PCR amplified from human genomic DNA (HEK293 cells) with the following primer pair: FP-miR-25: 5′-gcggccgccattctcagacgtgcctaag-3′, RP-miR-25: 5′-tctagatgattacc-caacctactgct-3′. After sub-cloning the hsa-Pri-miR-25 amplicon that contains endogenous 5′- and 3′-flanking sequences was finally cloned into the pcDNA3.1(−) vector (Invitrogen) via BamHI (vector and insert) and NheI (vector) XbaI (insert). HPLC grade human synthetic Pre-miR™ precursor miRNAs that are strand-selection optimized/approved and chemically modified siRNA-like precursor miRs (Pre-miR-1™ #AM17100; Pre-miR-25™ #AM17100; Pre-miR-613™ #AM17100) were purchased from Ambion. Three Axin1 and Axin2 Stealth siRNA (Invitrogen, Cat. No.: 119026-B03, -B05, -B01, -007, -C11, -009) were used as positive control and epistatic-inductors to activate the Wnt pathway at its downstream components. Three Stealth siRNAs for β-catenin (Invitrogen, 119026B07/B09/B11) were used to down-regulated Wnt activity as positive control for a Wnt-pathway inhibitory RNA. Silencer-Negative Control siRNA #4 (Ambion, Invitrogen #AS00K2L1) and Silencer-Negative control siRNA #2 (Ambion, Invitrogen #AS00JSIG) were used as negative controls. The Pre-miR™ precursor molecule library was plated in 5 μL with 1.5 pmol each well into two 384 well plates at the RNAi screening core facility using an automated dispenser system (Janus MDT, Perkin Elmer) and stored at −80° C. pLV-miR-1 and pLV-miR-control lentivirus were obtained from Biosettia (San Diego, USA).
Bioinformatics, and Statistical Analysis
Sequences were aligned with ClustalW, visualized and processed with Genedoc software, and Treepuzzle and Treeview software were employed for similarity and phylogenetic analysis (parameters: 10.000 puzzling steps; quartet puzzling tree reconstruction, neighbor-joining tree, approximate quartet likelihood, HKY-model of substitution). miRNA target prediction and alignment blasts were done with miRWalk, Pictar, Diana-microT, PITA, RNAhybrid, Target Scan, miRanda, NCBI-blasts. Statistical analysis: Z-factors were calculated with Z=1−(3(σp+σn)/(|μp−μn|)) and values between 0.5 and 1.0 indicated good screening parameters. Log-Z-score was deployed on log-tansformed (Nexp FF/RL) data set. Z=((FF/RL)LOG−PALOG)/STDEV(PLOG); FF, TCF19x-firefly RLU; RL, CMV-Renilla RLU; PA, plate average; RLU, relative light units; STDEV(P) standard deviation of the plate. Local plate averages (7-10 data points) were used for alternative balancing of normalized data of the primary screen. Regulation of transcript level/translation is measured by the psi-check-2 reporter and determined by relative changes of RLU values via R(r)=(RL−β−catCDS/FF)/(RL−empty/FF); with R(r) of controls=1. Pubmed (NCBI) and Google searches were used for data mining to find cancer relevant reports on miRs identified and verified for correlation studies.
Western Blotting
SDS-PAGE and western blotting was performed with standard protocols including TBST (0.1%) washing buffer and 4% BSA TBST blocking buffer. Primary antibodies (anti-β-catenin, Sigma, #C7207, anti-tubulin (α-Tubulin), Sigma, #T9026) were incubated over night while gently shaking at 4° C. in 1:1000 to 1:2000 dilutions. Infrared (IR)-dye conjugated secondary antibodies (1:20.000, goat anti-mouse, IRDye™800, #610-132-121, Rockland) were incubated at room temperature for 1 h in blocking buffer and subsequently rinsed with TBST. Blots were visualized with the Odyssey infrared imaging system and quantified with the Odyssey software (Li-Cor, Biosciences). For semi-quantitative western blotting the average of the ratios of β-catenin and α-Tubulin (α-TUB) [signal intensity] was divided by the average of the ratios of the control experiments.
Cell Culture and Cell Lines
HEK293 human embryonic kidney cells (ATCC, cat # CRL-1573), HCT116 human colon cancer cells (ATCC, cat # CCL-247), SW480 human colon cancer cells (ATCC, cat # CCL-228), and MCF7 human breast cancer cells (ATCC, cat # HTB-22) were cultured in filtered DMEM media supplemented with 10% fetal calf serum, 1 mM L-glutamine and 1× non-essential amino acids (ATCC, #203166) without antibiotics at 37° C. and 5% CO2. Selection media for cells transfected with linearized empty pcDNA3.1(−) or Pri-hsa-miR-25 pcDNA3.1(−) using Lipofectamine2000 contained increasing amounts of active G418 sulfate (Cellgro #30-234-CR) for 7-16 days. Lenti-pLV-miR-1 and -pLV-control transduced cells were selected with puromycin for up to 7 days.
RT-qPCR Real-Time relative quantification PCR was conducted with 25 μL iTAQ™ SYBR-Green Supermix (lx) with ROX (BIO-RAD, #20361) and the qPCR thermocycler Mx3005P (Stratagene) system including the MxPro-Mx30055 v.4.10 Build 389 software (Stratagene) and the delta-delta-Cr calculation method. Thermocycler conditions: 96° C. initial denaturation step for 10 min, 50 cycles of (96° C. denaturation for 30 s, 57° C. annealing for 30 s and 72° C. elongation for 10 s). Amplification and dissociation curves revealed the specificity of the qPCR products, which were also examined by DNA agarose TAE gel electrophoresis (2% agarose, with EtBr). Unmodified exon-spanning primer pairs with similar annealing temperature (57° C.) and product size (80-110 bp) were used. Total RNA was isolated with RNeasy (Qiagen) and cDNA was synthesized using the High-capacity cDNA reverse transcription kit (Applied Biosystems, #4368814). Briefly, 1 μg of total RNA was DNaseI digested, heat inactivated, and input for cDNA reverse transcription in 20 μL following the instructions of the manufacturer. Primer sequences: CTNNB1-fw 5′-ATGGCAACCAAGAAAGCAAG-3′; CTNNB1-ry 5′-GGTCCACAGTAGTTTTTCGTAAG-3 (product size 101 bp); internal control primer: GAPDH-fw 5′-TGAAGGTCGGAGTCAACG-3′, GAPDH-ry 5′-GGGTCATTGATGGCAACA-3′ (product size 97 bp).
In Vivo Context Analysis of the Regulation of Axin2/Conductin-lacZ Reporter by miR-1:
Two axin2/Conductin-lacZ mature female mice [37] were sacrificed and mammary epithelial organoids prepared essentially as described [38]. Six mammary glands/mouse were collected in DMEM/F12 10% FBS medium on ice. The glands were transferred to a sterile Petri dish and minced into a homogenous paste using a pair of sterile scalpels. The minced tissue was transferred to a 15 ml falcon tube containing 9 ml Epicult-B Basal Medium (Stem Cell Technologies) and 1 ml of 10× collagenase and hyaluronidase (3000 and 1000 units/ml Stem Cell Technology) and constantly agitated (environmental shaker) for 1 h at 37° C. The tissue was then collected by centrifuged at 450 g for 10 min and re-suspended in cold HBSS 2% FBS. The organoids were isolated from contaminating fibroblasts and blood cells by 2 s pulse centrifugation at 450 g (repeated 5-7 times) their purity was assessed by examination on a glass slide. The final pellet was re-suspended in 2 ml Trypsin/EDTA (0.25%) and incubated at 37° C. for 1 min. The sample was then diluted in 10 ml of cold HBSS containing 2% FBS and spun down at 450 g for 5 min. The supernatant containing stringy DNA was removed carefully and the remaining pellet was re-suspended in pre-warmed fresh dispase containing 500 μl (2000 unit/ml) DNase, mixed for 1 min and spun down. Organoids were then re-suspended and plated in Epicult-B Basal Medium. Control and miR-1 expression vectors (pLV-miR-1 from Biosettia Inc., USA) were used for lentiviral transduction of the cells in 24-well plates. After 12 h of incubation, the cells were fixed in 4% formaldehyde (Electron Microscopy Sciences) for 20 min, washed 3 times with 1×PBS, permeabilized and blocked with blocking buffer (0.1% Triton X-100, 1% BSA, 5% normal goat serum in DPBS) at room temperature for 20 min and immuno-stained with mouse anti-β-GAL 1:50 (DHSB, 4° C. overnight), anti-mouse IgG-Alexa488 goat antibody (Invitrogen; 1:1000; 2 h at room temperature) and DAPI. The cells were imaged using a Nikon TE2000PFS and the levels of β-gal (fluorescence intensity) was measured by the NIS elements software (Nikon, USA).
Identification of Human microRNAs in a Primary Wnt-Reporter Screen
Micro-RNAs (miRNAs, miRs) and the Wnt pathway play fundamental roles in development and disease by regulating expression of target genes. We hypothesized that certain miRs could fine-tune the activity of β-catenin-dependent Wnt signaling by modulating the abundance of relevant pathway components. To address this question we performed a luciferase reporter screen in human HEK293 cells to investigate the regulatory capacity of a library of 470 miRs on the activity of the Wnt pathway. HEK293 cells were transiently co-transfected with individual synthetic Pre-miR™s, a luciferase gene reporter driven by 16 functional TCF/LEF1 binding sites and a CMV-Renilla luciferase that served as internal control for cell viability and transfection efficiency. The Wnt pathway was activated with Wnt3a-conditioned media from L-cells 2.5 days post-transfection, and plates were read after 16 h of Wnt3a-CM treatment at day 3. The primary screening was performed in quadruplets in a 384-well plate format and yielded a set of 60 (of 470) candidate Wnt-regulatory human miRNAs (30 activators/synergists and 30 repressors), representing 12.8% of the strand-selective, synthetic Pre-miR™ Library (Ambion) (
Wnt-Modulation by miRs Correlates with miR-Oncogenicity and Sequence Similarity.
Having determined the initial list of verified Wnt-modulating miRs (
Interestingly, further data mining via literature searches revealed another outstanding and surprising coherency that also supported our initial hypothesis: a majority of the miRs that have been reported to be anti-oncogenic and are repressed in cancers displayed Wnt-inhibitory properties, whereas those described as oncogenic miRs and are often elevated in cancers were identified as activators of the Wnt pathway by trend (
Secondary Validation of miR-1, miR-25 and miR-613.
We chose to further investigate three candidate miRs that repressed Wnt3a/β-catenin signaling in the primary screen: miR-1, miR-25 and miR-613. Phylogenetic analysis support the miR-base classification that miR-1 belongs to the miR-1/206 family including hsa-miR-206 and the Drosophila dme-miR-1, an indication of the high evolutionary conservation of this family (shown in
To understand at which step these miRs (miR-1, -25, -613) modulate the linear cascade of the Wnt pathway, and to identify their potential target genes, a series of epistasis experiments were conducted in HEK293 cells using the Wnt reporter and different pathway activators (
Relative quantification with real time quantitative PCR (RT-qPCR) did not reveal any significant reduction in β-catenin (CTNNB1) mRNA level in HEK293 cells (
The epistasis experiments indicated that miR-25 may act in parallel or downstream of β-catenin itself. Intriguingly the most stringent RNAhybrid predictions that allow non-canonical seed sequences indicated some potential binding sites of miR-25 in the β-catenin cDNA (
Investigating Functions of miR-1 and miR-25 in Wnt-Relevant Colon Cancer Cell Lines.
In order to investigate the function of miR-25 in Wnt-responsive cell lines we cloned a human unprocessed Pri-miR-25 into the pcDNA3.1(−) expression vector with a selectable neomycin marker. Upon transfection of colon cancer cells (HCT116, HT29, SW480) with Pri-miR-25 expressing vector, the number of Pri-miR-25 stable cell-colonies was markedly reduced compared to empty vector controls (
We also investigated the potential function of the candidate Wnt-inhibitor miR-1 in the Wnt-dependent HT29 cancer cell line, because miR-1 was identified as one of the strongest repressors of Wnt-3a-induced activation of the STF reporter (
miR-1 Inhibits Expression of a Wnt-Responsive Reporter (Conductin-lacZ) in Primary Mammary Organoids.
To test whether candidate miRs can influence Wnt signaling in an in vivo context we derived primary mammary epithelial organoids from the axin2/conductin-lacZ mouse [37] using protocols described in Teissedre et al. [38]. Conductin-lacZ has been previously shown to respond to activated-Wnt signaling in mammary epithelial tissue. We introduced a miR-1 expression construct into mammary epithelial organoids derived from the conductin-lacZ in vivo reporter mouse using lentiviral transduction (pLV-miR-1 from Biosettia Inc., USA) and investigated whether expression of miR-1 could influence the expression of the β-gal reporter compared to pLV-empty vector control. As shown in
In this report we provide the results from a comprehensive screen for the identification, and validation/characterization of human Wnt pathway-modulating miRNAs using a systematic HTS approach. Three candidate Wnt-repressing miRs, namely, miR-1, miR-25 and miR-613, were further characterized in cell-biological assays. In addition to the known Wnt-modulatory miRNAs, such as miR-200a [29], [32], Drosophila miR-315 [28], miR-8 [30], miR-27 [31], zebrafish miR-203 [33], and miR-34 [34], we identified 37 additional miRs that modulate the activity of the Wnt-pathway reporter in cultured human cells. The functions of these 37 miRs, their potential evolutionary conservation, as well as their putative target genes can now be addressed in future studies.
Notably, we uncovered an interesting correlation between many of the candidate miRs that exhibit sequence similarities, both within and outside their mature seed sequence, and their ability to exert similar modulating influence on the activity of the Wnt3a/β-catenin pathway, thereby indicating sequence specificity in miR-mediated Wnt-pathway modulation. The consensus sequences may indicate novel functional seed and “co-seed” sequences that may be involved in the modulation of the Wnt pathway. The partly disrupted consensus seed and co-seed sequences might reflect the imperfect base-pairing with their cognate target genes that participate in Wnt signaling. Moreover, alignment of miRs that could regulate the Wnt reporter made intra- and inter-family related functional consensus sequences apparent (i.e. the seed of miR-1 and miR-613 or within the miR-302 and -515 families (
Interestingly, our data also revealed that Wnt-inhibitory miRs tend to be anti-oncomiRs and Wnt-activating miRs tend to be oncomiRs. While the target-directness of each miR needs to be identified and validated in future studies, these data suggest that oncomiRs could contribute to elevated Wnt-pathway activity in cancers (
3 out of 38 candidate miRs (miR-1, miR-25, miR-613) were further characterized in Wnt-responsive cultured cells and all were validated for their Wnt-inhibitory properties identified in the initial screen. Epistasis experiments revealed that candidate miRs target the signaling network at different sites. Pre-mir-1 may function most upstream, followed by miR-613 and then miR-25, which seems to influence the most downstream activity at the level of J3-catenin. All miRs down-regulated Wnt3a-CM and LiCl induced Wnt pathway activity, while only Pre-miR-25 was able to repress Axin1+2-siRNAs or β-catenin-S37A induced activity. Hsa-Pre-mir-1 had a lesser ability to reduce total β-catenin protein levels under conditions of high pathway activation with LiCl. Interestingly, the results from this set of epistasis experiments are in agreement with a known function of Axin downstream/independent of the GSK3 β-catenin destruction complex (
Analyses of miR-25 function in the regulation of the Wnt pathway suggests a potential function in the translational inhibition of β-catenin via its binding to the β-catenin coding sequence and not its 3′-UTR. Expression of miR-25 repressed the psi-check2 sensor containing the miR-25 binding site, and moderately reduced β-catenin protein levels, while β-catenin transcript levels remained unchanged. Curiously the effect of hsa-miR-25 on β-catenin seems to be more effective under conditions of high pathway and low destruction complex activity, when translational differences preponderate and come into play due to strongly reduced post-translational regulation of β-cat. Reduced β-cat protein amounts can thus be better resolved in LiCl-induced HEK293 cells with high pathway activity, while a low pathway activity by Wnt-3a-CM (ca. 10-fold in STF assay, LiCl ca.50-100-fold) could only be reduced by miR-1 and miR-613 that can block a more upstream part of the pathway and are thus more efficient (
Finally, the very strong anti-proliferative effect of hsa-miR-1 in Wnt/β-catenin dependent human cancer cells (HT29) but not in HEK293 cells, combined with its strong inhibitory effect on an in vivo Wnt-reporter in primary mammary epithelial organoids (
In summary our study reports the first comprehensive identification of Wnt-modulating miRs in human cells and represents how miR-based HTS can be employed as a powerful tool to systematically identify pathway relevant miRs. Since pathway-modulatory miRs could have functional impact in cancers associated with deregulated cell signaling, these findings could also benefit the long-term goal of developing miR-based therapeutics and for the diagnostic classification of cancers by expression profile signatures.
This application claims priority under 35 USC §119(e) from U.S. Provisional Application Ser. No. 61/627,628, filed Oct. 14, 2011, which application is herein specifically incorporated by reference in its entirety.
The research leading to the present invention was supported, at least in part, by a Department of Defense (DOD)/BCRP concept award W81XWH-07-1-0541 and DOD-BC093088. Accordingly, the Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61627628 | Oct 2011 | US |
