Patent Application
20140356459
The invention relates to molecular targets and their use to counteract tumors. Naturally occurring microRNAs that regulate human oncogenes and methods of use thereof are described. Suitable nucleic acids for use in the methods and compositions described herein include, but are not limited to, pri-miRNA, pre-miRNA, mature miRNA or fragments of variants thereof that retain the biological activity of the mature miRNA and DNA encoding a pri-miRNA, pre-miRNA, mature miRNA, fragments or variants thereof, or regulatory elements of the miRNA. The here claimed approach is efficacious also on Cancer Stem Cells.
Cancer is a group of diseases characterized by uncontrolled growth and spread of abnormal cells. Cancer is caused by both external factors (tobacco, chemicals, radiation, and infections organisms) and internal factors (inherited mutations, hormones, immune conditions, and DNA damage). These factors may act together or sequentially to initiate and/or promote carcinogenesis. Cancer causes 1 of every 4 deaths and it is the leading cause of death in people under age 85 in the United States. Nearly half of all men and a little over one third of all women in the U.S. will develop cancer during their lifetimes. Today, millions of people are living with cancer or have had cancer. The sooner a cancer is found and treatment begins, the better are the chances for living for many years.
Misregulation of genes that control cell fate determination often contributes to cancer. Such altered genes are known as oncogenes. Oncogenes are called proto-oncogenes when they are normal (i.e., not mutated). Proto-oncogenes encode components of the cell's normal growth-control pathway. Some of these components are growth factors, receptors, signaling enzymes, and transcription factors.
In 2001, several groups used a novel cloning method to isolate and identify a large group of “microRNAs” (miRNAs) from C. elegans, Drosophila, and humans (Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001). Several hundreds of miRNAs have been identified in plants and animals—including humans—which do not appear to have endogenous siRNAs (small interference RNA). Thus, while similar to siRNAs, miRNAs are nonetheless distinct. miRNAs are 21-24 nucleotide (nt) duplex RNAs that are created from precursor transcripts by subsequent processing steps mediated by members of the RNAseIII family, Drosha and Dicer. One of the two strands is incorporated into the active sites of the Argonaute family of proteins, where it serves as a guide for WatsonCrick base pairing with complementary sequences in target messenger RNAs (mRNAs).
Animal miRNAs are initially transcribed as part of one arm of an ˜80 nucleotide RNA stem-loop that in turn forms part of a several hundred nucleotides long miRNA precursor termed a primary miRNA (pri-miRNA)s. The double-stranded RNA structure of the hairpins in a pri-miRNA is recognized by a nuclear protein known as DGCR8. DGCR8 associates with the enzyme Drosha to form a complex where DGCR8 orients the catalytic RNase III domain of Drosha to liberate hairpins from pri-miRNAs. The product resulting is often termed as a pre-miRNA (precursor-miRNA). A single pri-miRNA may contain from one to six precursor-miRNA. pre-miRNA hairpins are exported from the nucleus in a process involving the nucleocytoplasmic shuttle Exportin-5. This protein, a member of the karyopherin family, recognizes a two-nucleotide overhang left by the RNase III enzyme Drosha at the 3′ end of the pre-miRNA hairpin. In cytoplasm, the pre-miRNA hairpin is cleaved by the RNase III enzyme Dicer. This endoribonuclease interacts with the 3′ end of the hairpin and cuts away the loop joining the 3′ and 5′ arms, yielding an imperfect miRNA:miRNA* duplex about 22 nucleotides in length.
In mammals, the majority of miRNAs guide the RNA-induced silencing complex (RISC) to the 3′ untranslated regions (UTRs) of mRNA targets, with the consequence that translation of the target mRNAs is inhibited. miRNA molecules interrupt translation through precise or imprecise base-pairing with their targets. Although either strand of the duplex may potentially act as a functional miRNA, only one strand is usually incorporated into the RNA-induced silencing complex (RISC) where the miRNA and its mRNA target interact.
The roughly 1000 miRNAs identified until now are thought to regulate over 30% of the expressed mRNA (Croce, C. M. Causes and consequences of microRNA disregulation in cancer. Nat Rev Genet 10, 704-714, 2009). This explains why miRNAs play roles in many fundamental biological processes such as cell proliferation, apoptosis, stress response, development and differentiation. Hence, it is not surprising that a growing number of studies describe aberrant expression of miRNAs with oncogenic potential in many human tumors (Esquela-Kerscher, A. & Slack, F. J. Oncomirs—microRNAs with a role in cancer. Nat Rev Cancer 6, 259-269, 2006). The connection between aberrant miRNA expression and tumor growth, spread and survival, suggests a new strategy to fight cancer by interfering with miRNA function. This strategy is particularly interesting, as the current technology allows for RNA-based pharmacological approaches not only in vitro, but also in vivo. Recent studies demonstrate a functional modulation of miRNA- and mRNA-levels using antagomiR-, LNA- and siRNA-treatments in mice (Ma, L., et al. Therapeutic silencing of miR-10b inhibits metastasis in a mouse mammary tumor model. Nat Biotechnol, 2010), non-human primates (Elmen, J., et al. LNA-mediated microRNA silencing in non-human primates. Nature 452, 896-899, 2008; Lanford, R. E., et al. Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection. Science 327, 198-201, 2010) and even in humans (Davis, M. E., et al. Evidence of RNAi in humans from systemically administered sRNA via targeted nanoparticles. Nature, 2010). These results strongly suggest that pharmacological manipulation of miRNA expression might represent a functional instrument for innovative anti cancer therapy.
Conventional anti cancer therapies such as radiation and chemotherapy mainly aim to kill fast proliferating tumor cells by inducing DNA damage or interfering with mitosis. However, unfortunately they show only limited efficacy in the treatment of solid tumors like breast, lung and colorectal cancer. For solid malignancies these widely accepted clinical approaches may delay the progression of the disease, but very rarely lead to complete eradication of the tumor. This poor clinical outcome as well as the marked heterogeneity observed in many solid tumors can be explained by the existence of Cancer Stem Cells (CSCs). CSCs represent an undifferentiated pool of cells within the tumor mass, which have high self renewal capacities and are responsible for tumor growth and spread (Clarke, M. F., et al. Cancer stem cells--perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res 66, 9339-9344, 2006). They were shown to be highly chemo- and radio-resistant and therefore are likely to be responsible for the frequent relapses after conventional tumor treatment observed in clinics. CSCs isolation and characterization has been achieved in many solid tumors on the basis of phenotypic and functional features and still represents a field of intensive investigation (Visvader, J. E. & Lindeman, G. J. Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat Rev Cancer 8, 755-768, 2008). The recent identification of CSCs from solid tumors has generated new perspectives for the development of cancer therapies potentially able to eradicate the entire neoplastic population.
The present study identifies a defined series of molecular targets, in particular miRNAs, involved in the proliferation of cancer cells and, preferably, of CSCs, thus offering a novel therapeutic approach in tumors.
The present invention identifies specific and new molecular target to be addressed to achieve an efficacious therapeutic outcome in tumors therapy. A genome-wide anti-miRNA screenings has been performed, systematically neutralizing the function of a given hsa-miRNA (Homo sapiens miRNA) in primary breast and lung cancer derived CSCs. The obtained results were then confirmed in commercially available cell lines. To neutralize more than 90% of experimentally confirmed hsa-miRNAs expressed by human cells, a comprehensive locked nucleic acids (LNA) based anti-miRNA library has been used. This library consists of LNAs targeting more than 800 miRNAs (miRCURY LNA™ microRNA inhibitor library, Human v.12.0, Exiqon). LNA-mediated miRNA neutralization has been chosen as this technology has a number of advantages compared to standard anti-miRs or antagomiRs:
By a genome-wide anti-miRNA screening, a series of miRNA to be targeted in cancer therapy via available technologies has been identified.
What we claim are hsa-miRNA to be inhibited to achieve a favorable therapeutic outcome in tumors, in particular in breast and lung tumors. The identified hsa-miRNA are referred to by using the standard nomenclature system and they are: hsa-miR-133a (SEQ. ID 1)133b (SEQ. ID 2), hsa-miR-1271 (SEQ. ID 3), hsa-miR-361-3 (SEQ. ID 4), hsa-miR-1226* (SEQ. ID 5), hsa-miR-512-5p (SEQ. ID 6), hsa-miR-1285 (SEQ. ID 7), hsa-miR-197 (SEQ. ID 8), hsa-miR-Let-7b* (SEQ. ID 9), hsa-miR-346 (SEQ. ID 10), hsa-miR-1252 (SEQ. ID 11), hsa-miR-615-3p (SEQ. ID 12), hsa-miR-636 (SEQ. ID 13), hsa-miR-1227 (SEQ. ID 14), hsa-miR-885-5p (SEQ. ID 15), hsa-miR-766 (SEQ. ID 16), hsa-miR-1296 (SEQ. ID 17), hsa-miR-204 (SEQ. ID 18), hsa-miR-191 (SEQ. ID 19), hsa-miR-663 (SEQ. ID 20), hsa-miR-574-3p (SEQ. ID 21), hsa-miR-342-3p (SEQ. ID 22), hsa-miR-92a (SEQ. ID 23), hsa-miR-486-5p (SEQ. ID 24), hsa-miR-150 (SEQ. ID 25), hsa-miR-210 (SEQ. ID 26), hsa-miR-621 (SEQ. ID 27), hsa-miR-145 (SEQ. ID 28), hsa-miR-1229 (SEQ. ID 29), hsa-miR-596 (SEQ. ID 30), hsa-miR-296-5p (SEQ. ID 31).
The inhibition of the activity of hsa-miR-133a133b, hsa-miR-1271, hsa-miR-361-3, hsa-miR-1226*, hsa-miR-512-5p, hsa-miR-1285, hsa-miR-197, hsa-miR-Let-7b*, hsa-miR-346, hsa-miR-1252, hsa-miR-615-3p, hsa-miR-636, hsa-miR-1227, hsa-miR-885-5p, hsa-miR-766, hsa-miR-1296, hsa-miR-204, hsa-miR-191, hsa-miR-663, hsa-miR-574-3p, hsa-miR-342-3p, hsa-miR-92a, hsa-miR-486-5p, hsa-miR-150, hsa-miR-210, hsa-miR-621, hsa-miR-145, hsa-miR-1229, hsa-miR-596, hsa-miR-296-5p is obtained by one of the available technologies, preferably selected from a locked nucleic acid (LNA) oligo, a Morpholino oligo or a 2′-O-methyl RNA oligo. Moreover, said at least one miRNA is silenced by a complementary antagomirR or specific steric-blocking oligo that inhibit the maturation of said miRNA. In a second embodiment, transcriptional inhibition of at least one of said miRNAs is obtained through sRNA directed against the genomic miRNA promoter, thus inducing chromatin changes, such as histone acetylation. In a third embodiment, the amount of processed miRNA available for target gene silencing is reduced with sRNA directed against the miRNA biogenesis components such as Drosha.
Said inhibition of at least one of a hsa-miRNA selected from hsa-miR-133a133b, hsa-miR-1271, hsa-miR-361-3, hsa-miR-1226*, hsa-miR-512-5p, hsa-miR-1285, hsa-miR-197, hsa-miR-Let-7b*, hsa-miR-346, hsa-miR-1252, hsa-miR-615-3p, hsa-miR-636, hsa-miR-1227, hsa-miR-885-5p, hsa-miR-766, hsa-miR-1296, hsa-miR-204 has a cytotoxiccytostatic effect on both cancer stem cells and differentiated carcinoma cells, preferably from breast or lung tumors.
Said inhibition of at least one of the hsa-miRNA selected from hsa-miR-574-3p, hsa-miR-342-3p, hsa-miR-92a, hsa-miR-486-5p, hsa-miR-150, hsa-miR-210, hsa-miR-621, hsa-miR-145, hsa-miR-1229, hsa-miR-596, hsa-miR-191, hsa-miR-296-5p, hsa-miR-663 leads to a cytotoxiccytostatic effect specifically on differentiated cancer cells, preferably the effect is achieved on breast tumor epithelial cells, non-small-cell lung carcinoma cells, lung tumor epithelial cells.
In a further embodiment of the present invention, we claim a combination with conventional chemotherapeutic drugs of at least one of the inhibitors of miRNA hsa-miR-133a/133b, hsa-miR-1271, hsa-miR-361-3, hsa-miR-1226*, hsa-miR-512-5p, hsa-miR-1285, hsa-miR-197, hsa-miR-Let-7b*, hsa-miR-346, hsa-miR-1252, hsa-miR-615-3p, hsa-miR-636, hsa-miR-1227, hsa-miR-885-5p, hsa-miR-766, hsa-miR-1296, hsa-miR-204, hsa-miR-191, hsa-miR-574-3p, hsa-miR-342-3p, hsa-miR-92a, hsa-miR-486-5p, hsa-miR-150, hsa-miR-210, hsa-miR-621, hsa-miR-145, hsa-miR-1229, hsa-miR-596, hsa-miR-191, hsa-miR-hsa-296-5p, hsa-miR-663. Preferably, said chemotherapeutic drugs are paclitaxel and/or cisplatin. One of said combinations is preferably used to treat breast or lung tumors.
We further claim a set of miRNA having a tumors suppressor function. These tumors suppressors hsa-miRNA are: hsa-miR-206 (SEQ. ID 32), hsa-miR-422a (SEQ. ID 33), hsa-miR-33a (SEQ. ID 34), hsa-miR-330-5p (SEQ. ID 35), hsa-miR-526b (SEQ. ID 36), hsa-miR-383 (SEQ. ID 37), hsa-miR-1282 (SEQ. ID 38), hsa-miR-101* (SEQ. ID 39), hsa-miR-548 family (SEQ. ID 40), hsa-miR-520 family (SEQ. ID 41), hsa-miR-370 (SEQ. ID 42). The tumor suppressor activity of the above listed miRNA is in particular observed in breast cancer. A transient increased expression of at least one of said miRNA is accomplished through the introduction of a synthetic miRNA mimic into the cytoplasm that can be processed and loaded into miRNA RNA induced silencing complex (miRISC) by Dicer. This is an approach of choice for local administration in easily accessible tissues, such as lungs. In a different approach, miRNAs is expressed from RNA polymerase (Pol) III promoters as an artificial short hairpin RNA (shRNA) that bypasses Drosha processing, yet is cleaved and loaded into miRISC by Dicer. Alternatively, the entire pri-miRNA is expressed from an RNA Pol II promoter, leaving open the possibility for tissue-specific or induced ectopic miRNA expression.
Moreover, hsa-miR-548 family, hsa-miR-520 family, hsa-miR-208a (SEQ. ID 43) and hsa-miR-208b (SEQ. ID 44), hsa-miR-545-3p (SEQ. ID 45) and hsa-miR-545-5p (SEQ. ID 46), hsa-miR-196b (SEQ. ID 47) are capable to avoid the resistance of tumor cells to standard chemotherapeutic agents. Preferably, an increased functionality of hsa-miR-548 family, hsa-miR-520 family, hsa-miR-208a and hsa-miR-208b, hsa-miR-545-5p and hsa-miR-545-3p, hsa-miR-196b prevents paclitaxel resistance in breast cancer and cisplatin resistance in lung cancer.
The detailed description of the embodiments and the examples that are reported below are given by way of illustration and are not intended to limit the present invention.
(i)133a and 133b belong to the same family. When results are indicated as 133a/133b, the reported values are the mean result of the two mature miRNAs
(ii)when miR-548 family is indicated, the reported values are the mean result of all the mature miRNAs belonging to the family.
(iii)when miR-520 family is indicated, the reported values are the mean result of all the mature miRNAs belonging to the family.
(iv)208a and 208b belong the same family. When results are indicated as 208a/208b the reported values are the mean result of the two mature
(v)545-3p and 545-5p belong the same family. When results are indicated as 545-3p/545-5p the reported values are the mean result of the two mature miRNAs.
Breast cancer stem cells, lung cancer stem cells derived from patient samples and commercially available breast and lung cancer cell lines were seeded in 96 well, plates and transfected with the LNA-based anti-miR 190101-00-miRCURY LNA™ microRNA Inhibitor Library—Human v. 12.0 using a lipofection reagent (Dharmafect 2 [Dharmacon] or, alternatively, HiPerfect [Qiagen]).
The transfection efficiency was controlled by a siRNA mediated knockdown of an indicator gene (NF-1B) followed by the analysis of NF-1B mRNA levels 48 hours post transfection using quantitative PCR. The chosen transfection conditions led to knockdown rates of 85-95% for NF-1B, indicating an efficient uptake of siRNA oligonucleotides under the screening conditions (
72-96 hours after transfection, cell viability was determined using an ATP-based readout (CellTiter-Glo [PROMEGA]). The relative viability was calculated by defining the average of the plates as 100% viability. This value was used to normalize the individual values obtained in the single wells.
A number of LNAs have been found to influence the viability of breast and lung cancer stem cells, as well as differentiated cell lines from both entities in both directions, leading to a decrease, or to an increase in cell number after treatment (
18 LNAs that led to a marked decrease of cell viability in cells transfected with the inhibitor have been identified (see table 1). Most of these LNAs showed pharmacological efficiency in all cellular systems analyzed (breast cancer stem cells, differentiated breast cancer line MDAMB231, lung cancer stem cells (LCSC136) and the lung cancer lines NIH-H460 and A549).
These data indicate that the identified specific inhibitors are able to reduce the viability of all tumor cells tested, including primary cancer stem cells. Thus, these LNAs either neutralize miRNAs or interfere with other pathways important for the general tumor cell survival, at least in vitro. The LNAs depicted in table 1 represent valuable tools for targeted anti-cancer therapy, able to eradicate not only the bulk of differentiated tumor cells, but also drug resistant cancer stem cells, that might be responsible for the frequent relapses after chemotherapy observed in clinics.
In the same experimental conditions as above, a series of LNAs have been identified selectively killing all three differentiated cell lines tested but not cancer stem cells (see table 2).
The reported LNAs have a high therapeutic value, for example to debulk the main tumor mass as adjuvant treatment before surgery.
LNA-transfected cells were also treated with conventional chemotherapeutic drugs. All LNAs listed in table 1 and table 2 also support the efficiency of chemotherapeutic drugs such as paclitaxel and cisplatin, commonly used to treat patients with breast and lung cancer, respectively.
By applying the same experimental conditions as detailed in example 1, another set of LNAs have been identified as capable to promote the cell growth of the transfected cells. This indicate that the LNAs neutralized miRNAs might act as tumor suppressors. This effect was especially pronounced in the breast cancer stem cell line (BCSC208), in which 11 LNAs led to a significant increase of viability. The data are provided in table 3 that follows.
The above reported targeted miRNAs might represent tumor suppressor genes which, when overexpressed by using any of the available technologies, can lead to a decrease in proliferation or even to the death of the tumor cells.
The genome wide anti miRNA screening detailed in example 1 was then performed after exposure of the indicated cells to chemotherapeutic agents, paclitaxel or cisplatin. A series of LNAs making tumor cells resistant to chemotherapy induced cell death has been identified. These LNAs, listed in table 4, prevented cell death induced by paclitaxel and cisplatin in breast cancer and lung cancer cells, respectively.
AntagomiR is a synthetic short, single strand RNA conjugated to cholesterol that is perfectly complementary to the specific miRNA target. AntagomiRs are here used as a method to constitutively inhibit the activity of the specific miRNAs identified in the LNA-screens reported in examples 1-4. A cytostatic and cytotoxic activities have been observed with the antagomiR-197 and −663 in lung cancer stem cells (LCSC136) and differentiated lung cancer cell lines (A549 and NIH-H460), respectively. Moreover, antagomiR-361-3p was able to substantially induce cell death in breast cancer stem cell clones tested. In addition, antagomiR-361-3p and antagomiR-197 showed only minimal cytotoxic effects on normal, non-transformed cells isolated from breast- and lung tissue, respectively.
| Number | Date | Country | Kind |
|---|---|---|---|
| 11193873.4 | Dec 2011 | EP | regional |
| MI2011A002272 | Dec 2011 | IT | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2012/057176 | 12/11/2012 | WO | 00 | 6/13/2014 |
