miRNA-193a for Promoting Immunogenic Cell Death

Abstract

The invention relates to the use of miRNA-193a for regulating gene expression, particularly it relates to the use of miRNA-193a as a CRT agonist, promoting the cell surface expression of CRT. This allows the advantageous treatment of cancers without or with low surface expression of CRT. The invention further relates to compositions comprising the miRNA for use in such treatment.

Claims

1. A method for treating a condition associated with low expression of calreticulin (CRT) or with an impaired immunogenic cell death (ICD) pathway, the method comprising the step of administering a miRNA-193a or a source thereof, or composition comprising a miRNA-193a or a source thereof.

2. The method according to claim 1, wherein the miRNA-193a is a CRT agonist or promotes cell surface expression of CRT or rescues or restores the ICD pathway.

3. The method according to claim 1, wherein the miRNA-193a is a miRNA-193a molecule, an isomiR, or a mimic thereof.

4. The method according to claim 1, wherein a source of a miRNA is a precursor of a miRNA and is a nucleic acid of at least 50 nucleotides in length.

5. The method according to claim 1, wherein said miRNA shares at least 70% sequence identity with any one of SEQ ID NOs: 56, 121, or 122, and/or wherein said miRNA is from 15-30 nucleotides in length, and/or wherein said source of a miRNA is a precursor of said miRNA and shares at least 70% sequence identity with any one of SEQ ID NOs: 5 or 13.

6. The method according to claim 1, wherein the condition associated with low expression of CRT is a low-CRT cancer or a cancer wherein the ICD pathway is impaired.

7. The method according to claim 1, wherein the low-CRT cancer is a low-CRT sarcoma, brain cancer, head and neck cancer, breast cancer, lung cancer, kidney cancer, liver cancer, colon cancer, ovarian cancer, melanoma, pancreatic cancer, thyroid cancer, hamartoma, tumour of the haematopoietic and lymphoid malignancy, or prostate cancer.

8. The method according to any one of claims 1-7, wherein the miRNA-193a modulates expression of a gene selected from the group consisting of CRT, RPS6KB2, KRAS, PDGFRB, SOS2, TGFBR3, CASP9, INPPL1, PIK3R1, PTK2, CBL, PDPK1, CCND1, BCAR1, MAGI3, MDM2, YWHAZ, and MCL1, and HMGB1.

9. (canceled)

10. The method according to claim 1, wherein the composition further comprises a further miRNA or precursor thereof, wherein the further miRNA is selected from the group consisting of miRNA-323, miRNA-342, miRNA-520f, miRNA-520f-i3, miRNA-3157, and miRNA-7, or an isomiR thereof, or a mimic thereof.

11. The method according to claim 1, wherein the composition further comprises an additional pharmaceutically active compound.

12. The method according to claim 1, wherein the composition is a nanoparticle composition, the nanoparticle comprising a diamino lipid and the miRNA-193a or a source thereof, wherein the diamino lipid is of general formula (I) ##STR00004## wherein n is 0, 1, or 2, and T.sup.1, T.sup.2, and T.sup.3 are each independently a C.sub.10-C.sub.18 chain with optional unsaturations and with zero, one, two, three, or four substitutions, wherein the substitutions are selected from the group consisting of C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkenyl, and C.sub.1-C.sub.4 alkoxy.

13. The method according to claim 12, wherein the nanoparticles comprises: i) 20-60 mol % of diamino lipid, and ii) 0-40 mol % of a phospholipid, and iii) 30-70 mol % of a sterol, and iv) 0-10 mol % of a conjugate of a water soluble polymer and a lipophilic anchor.

14. An in vivo, in vitro, or ex vivo method for agonising CRT or for increasing cell surface expression of CRT, the method comprising the step of contacting a cell with a miRNA-193a or a source thereof, or with a composition comprising a miRNA-193a or a source thereof.

15. A method for treating a low-CRT cancer, the method comprising the step of administering to a subject a miRNA-193a or a source thereof, or a composition comprising a miRNA-193a or a source thereof.

16. The method according to claim 1, wherein the miRNA-193a is an oligonucleotide with a seed sequence comprising at least 6 of the 7 nucleotides of the seed sequence represented by SEQ ID NO: 22.

17. The method according to claim 8, wherein the miRNA-193a modulates expression of a gene selected from the group consisting of RPS6KB2, KRAS, PDGFRB, CASP9, INPPL1, PIK3R1, PTK2, CBL, PDPK1, CCND1, BCAR1, MAGI3, MDM2, YWHAZ, and MCL1.

18. The method according to claim 8, wherein the miRNA-193a modulates expression of a gene selected from PDPK1 and INPPL1.

19. The method according to claim 11, wherein the additional pharmaceutically active compound is selected from the group consisting of a PP2A methylating agent, an inhibitor of hepatocyte growth factor (HGF), an antibody, a PI3K inhibitor, an Akt inhibitor, an mTOR inhibitor, a binder of a T cell co-stimulatory molecule, and a chemotherapeutic agent.

20. The method according to claim 19, wherein the binder of a T cell co-stimulatory molecule is a binder of OX40.

Description

FIGURE LEGENDS

[0273] FIG. 1—Canonical pathway analysis. (A) Top 25 canonical pathways regulated by miR-193a in at least three cell lines at 24 h ranked based on P value. Dotted line indicates P<0.01. White bar: activated, black bar: inhibited, grey bar: direction not determined. (B) Treemap of genes that were identified in at least 4 significant pathways. Box size corresponds to the number of pathways in which the gene was differentially expressed.

[0274] FIG. 2—Genes downregulated by miR-193a in the PTEN pathway. Genes that were significantly downregulated by miR-193a (average relative expression compared to mock <1, P<0.05) at 24 h in at least three cell lines are shown without hatching. PTEN is highlighted in black. Pointy arrows indicate stimulation whereas bar-headed arrows indicate inhibition.

[0275] FIG. 3—Biological functions affected by miRNA-193a. Biological functions relevant to (tumour) cells with z-scores <−2 and >2. All P values are smaller than 0.00001.

[0276] FIG. 4—Western blotting of miR-193a-3p targets in the PTEN pathway. Human tumor cell lines were transfected with 10 nM scrambled control or 10 nM miRNA-193a and lysed after 72 h. Clarified whole cell lysates were immunoblotted for FAK, P70S6K, PIK3R1 and TGFBRIII. Vinculin and tubulin are loading controls. Boxes indicate protein downregulation.

[0277] FIG. 5—Western blotting of phosphoproteins in the PTEN pathway. Human tumor cell lines were transfected with 10 nM scrambled control or 10 nM miRNA-193a and lysed after 72 h. Clarified whole cell lysates were immunoblotted for pSer473 AKT, AKT, pThr202/Tyr204 ERK1/2, ERK1/2, pSer259 c-RAF and c-RAF. Vinculin and tubulin are loading controls. Full boxes indicate protein downregulation and dashed boxes indicate protein upregulation.

[0278] FIG. 6—Transfection with miRNA-193a induces surface expression of CRT in A2058, HEP3B, HCT116, and Huh7 cells. (A-B) Graphs show percentages of live (DAPI.sup.−) and dying (DAPI.sup.low), but not dead (DAPI.sup.+) A2058 and Hep3B cells, expressing CRT on their surface. Cells transfected with 0.1, 1, 3 and 10 nM of miRNA-193a, or a mock transfection control. (C-D) Panels show the cytofluorometric plots of A2058 (C) and HEP3B (D) cells, analyzed 72 hours post transfection. (E-F) Graphs show percentage of live (DAPI.sup.−) HCT116 and Huh7 cells expressing CRT on their surface, 96 h post transfection with 1 and 10 nM of miRNA-193a, or a mock transfection control. (G-H) Panels show the cytofluorometric plots of HCT116 (G) and Huh7 (H) cells, analyzed 96 hours post transfection.

[0279] FIG. 7—miRNA-193a-transfected cells are able to markedly stimulate maturation of dendritic cells from monocytes in co-culture assay. Monocytes were kept in culture co-culture with mock transfected or miRNA-193a transfected (1 or 10 nM) A2058 cells. Surface expression of CD80 and MHC class II molecules as markers of mature DC, were measured with flow cytometry, 7 days post co-culture. TNFα was used as a known DC maturation stimulator.

[0280] FIG. 8—Co-culture with miRNA-193a transfected A2058 tumor cells enhances the proliferation of T cells. PBMCs were labeled with CFSE and kept in culture alone, or in co-culture with mock transfected or miRNA-193a transfected A2058 cells. Cytofluorometric plots show the level of CFSE of CD3+ T cells, after 2 days or 6 days of co-culture.

[0281] FIG. 9—Effect of human Peripheral Blood Mononuclear Cells (PBMCs) on human melanoma A2058 and NSCLC A549 tumor cells. Human melanoma A2058 (A) and NSCLC A549 (B) tumor cells were co-cultured with the indicated ratio of human PBMCs to tumor cells for 72 h either in the absence or the presence of human anti CD3/CD28 antibodies (T cell activator). Then surviving cells were fixed and stained with crystal violet. The relative percentage of surviving cells (as compared to similar experimental conditions in the absence of PBMCs) was quantified by colorimetry of the stained cells (Feoktistova et al., 2016). Error bars represent SD to the mean of 3 independent replicates.

[0282] FIG. 10—Effect of human Peripheral Blood Mononuclear Cells (PBMCs) on human melanoma A2058 (A) and NSCLC A549 (B) tumor cells upon tumor cell transfection with miRNA-193a. Human melanoma A2058 and NSCLC A549 tumor cells were transfected (RNAiMAX) with the indicated concentrations of either negative miRNA control or miR-193a-3p, after which cells were co-cultured with the indicated ratio of human PBMCs to tumor cells for the indicated times. Then surviving cells were fixed and stained with crystal violet. The relative percentage of surviving cells (as compared to mock transfected condition)) was quantified by colorimetry of the stained cells (Feoktistova et al., 2016). N.S.: not significant, *: p<0.05 and **: p<0.01 as determined by the Student t tests (asymptotic significance [2-tailed]). Error bars represent SD to the mean of 3 independent replicates.

EXAMPLES

Example 1—RNA-Sequencing, Differential Gene Expression, and Pathway Analysis after Treatment of Different Cancer Cell Lines with miRNA-193a

[0283] Implementation of high-throughput RNA-sequencing (RNA-seq) has become a powerful tool for comprehensive characterization of the whole transcriptome at gene and exon levels and with a unique ability to identify differentially expressed genes, novel genes and transcripts at high resolution and efficiency. However, till date, very few miRNAs have been characterized for their specific role in cancer development. Hence, we have used the high-throughput RNA-seq after overexpressing a miRNA-193a (viz. a miRNA-193a-3p mimic) in 5 different cancer cell lines including A540 and H460 (both lung cancer), Huh7 and Hep3B (both liver cancer), and BT549 (breast cancer) at 24 h post-transfection with miR-193a at 10 nM. The gene expression was compared to mock as control and differentially expressed genes and their cellular pathways were subsequently identified.

1.1 Materials & Methods

[0284] 1.1.1 Cell Preparation for RNA-Sect

[0285] Human cancer cell lines were cultured in appropriate media (Table 1) and seeded into δ-well plates 24 h before transfection with 10 nM miRNA-193a-3p mimic or mock using Lipofectamine RNAiMAX (Thermofisher). The mimic was a double stranded mimic wherein the antisense strand consisted of an RNA oligonucleotide having SEQ ID NO: 56 (the canonical miRNA-193a-3p), and wherein the sense strand consisted of an oligonucleotide represented by SEQ ID NO: 218.

[0286] Reagents were aspirated 16 h after transfection and cells were passaged into new δ-well plates. Media was aspirated 24 h after transfection and plates were stored at −80° C. Three independent replicates were performed for each cell line.

TABLE-US-00001 TABLE 1 Cell line details. Cell line Cancer type Medium A549 Lung (NSCLC) F-12K + 10% FBS + P/S BT549 Breast (TNBC) RPMI-1640 + 10% FBS + P/S + 0.023 IU/mL insulin H460 Lung (NSCLC) RPMI-1640 + 10% FBS + P/S HEP3B Liver (HCC) EMEM + 10% FBS + P/S HUH7 Liver (HCC) DMEM low glucose + 10% FBS + P/S + L-glutamine FBS: fetal bovine serum, P/S: penicillin streptomycin

[0287] 1.1.2 RNA Isolation for RNA-Sect

[0288] RNA was isolated using the miRNeasy Mini kit (Qiagen). The procedure included on-column DNase treatment. RNA concentration was measured on NanodropOne. 150 ng of each independent replicate was pooled and 450 ng samples (having sample IDs A549 Mock_24, A549 miRNA-193a-3p_24, BT549 Mock_24, BT549 miRNA-193a-3p_24, H460 Mock_24, H460 miRNA-193a-3p_24, HEP3B Mock_24, HEP3B miRNA-193a-3p_24, HUH7 Mock_24, and HUH7 miRNA-193a-3p_24) were submitted to GenomeScan BV (Leiden, The Netherlands).

[0289] 1.1.3 RNA-Seq Procedure

[0290] PolyA enrichment was performed followed by next generation RNA-Seq using Illumina NovaSeq 6000 at GenomeScan BV. The data processing workflow included raw data quality control, adapter trimming, and alignment of short reads. The reference GRCh37.75.dna.primary_assembly was used for alignment of the reads for each sample. Based on the mapped locations in the alignment file the frequency of how often a read was mapped on a transcript was determined (feature counting). The counts were saved to count files, which serve as input for downstream RNA-Seq differential expression analysis.

[0291] 1.1.4 Data Analysis for RNA-Sect

[0292] Differential expression analysis was performed on the short read data set by GenomeScan BV. The read counts were loaded into the DESeq package v1.30.0, a statistical package within the R platform v3.4.4. DESeq was specifically developed to find differentially expressed genes between two conditions (mock versus miRNA-193a-3p) for RNA-seq data with small sample size and over-dispersion. The differential expression comparison grouping is provided in Table.

TABLE-US-00002 TABLE 2 Expression comparison setup. Comparison Condition A Condition B 1 A549_Mock_24 A549_miRNA-193a-3p_24 2 BT549_Mock_24 BT549_miRNA-193a-3p_24 3 H460_Mock_24 H460_miRNA-193a-3p_24 4 HEP3B_Mock_24 HEP3B_miRNA-193a-3p_24 5 HUH7_Mock_24 HUH7_miRNA-193a-3p_24

[0293] 1.1.5 Pathway Analysis

[0294] Lists of genes that were significantly (P<0.05) differentially expressed in our RNA-seq dataset were uploaded and analyzed using Ingenuity Pathway Analysis (IPA) software (www.ingenuity.com).

1.2 Results

[0295] 1.2.1 Genes Regulated by miR-193a-3p Mimic in Solid Tumor Cell Lines

[0296] Lists of significantly (P<0.05) differentially expressed genes (relative expression miRNA-193a/relative expression mock) at 24 h after transfection were created for all cell lines (see description of invention). Most genes were downregulated as compared to mock (relative expression miRNA-193a/relative expression mock <1) (see Table 3).

TABLE-US-00003 TABLE 3 Number of genes down- and upregulated by 193a-3p mimic per cell line. 24 h Down Up A549 615 220 BT549 620 168 H460 656 215 HEP3B 599 166 HUH7 683 200

[0297] Table 4 shows genes with known roles in cancer that were downregulated by miRNA-193a in each cell line. Genes that were downregulated in all cell lines include: CCND1, CDK6, KRAS, MCL1, NTSE, STMN1, TGFBR3 and YWHAZ.

TABLE-US-00004 TABLE 4 Genes of interest downregulated by miR-193a per cell line. Cel lines Downregulated genes A549 CAPRIN1, CCNA2, CCND1, CDK4, CDK6, CHEK1, DCAF7, DDB1, ETS1, HDAC3, HMGB1, IL17RD, KRAS, MCL, MPP2, NOTCH2, NT5E, PLAU, PSEN1, PTK2, RAB27B, SEPN1, SLC7A5, SOS2, ST3GAL4, STAT3, STMN1, TGFB2, TGFBR2, TGFBR3, TNFRSF21, YAP1, YWHAZ BT549 CCNA2, CCND1, CDC25A, CDK4, CDK6, CSF2, DCAF7, DDB1, ETS1, GRB7, HIC2, IDO1, IL17RD, KRAS, MCL1, MDM2, MPP2, NOTCH2, NT5E, PLAU, PSEN1, PTK2, RAB27B, SEPN1, SLC7A5, SOS2, ST3GAL4, STMN1, TGFBR3, TNFRSF1B, TNFRSF21, YAP1, YWHAZ H460 CAPRIN1, CCNA2, CCND1, CDK6, CDKN1A, CHEK1, CXCL1, CXCL5, DCAF7, DDB1, ETS1, HMGB1, IL17RD, KRAS, MAPK8, MCL1, MPP2, NOTCH1, NOTCH2, NOTCH3, NT5E, PLAU, PSEN1, PTK2, SEPN1, SLC7A5, ST3GAL4, STMN1, TGFBR3, TNFRSF1B, TNFRSF21 HEP3B AJUBA, CAPRIN1, CCND1, CDK6, CRYAA, DCAF7, ERBB4, ETS1, GRB7, IL17RD, KRAS, MAPK8, MCL1, MDM2, MPP2, NOTCH1, NT5E, PSEN1, PTK2, SEPN1, SLC7A5, SOS2, ST3GAL4, STMN1, TGFBR2, TGFBR3, TNFRSF1B, TNFRSF21, YAP1, YWHAZ HUH7 BRCA1, CCNA2, CCND1, CDC25A, CDK1, CDK6, CHEK1, DCAF7, E2F1, ETS1, EZH2, FEN1, FOXM1, GRB7, HMGB1, IL17RD, KRAS, MAPK8, MCL1, MDM2, MELK, MPP2, NT5E, PLAU, PLK1, RAD51, SEPN1, SLC7A5, ST3GAL4, STMN1, TGFBR3, TNFRSF21, YWHAZ

[0298] 1.2.2 Cellular Pathways Regulated by miR-193a in Solid Tumor Cell Lines

[0299] IPA was performed to identify canonical pathways that are affected in miRNA-193a treated cells compared to mock, based on the differential expression data. Because the objective was to develop new treatment options by more closely defining the mode of action of miR-193a across cancer types, we next analyzed the pathways that were regulated by genes differentially expressed in at least three cell lines. This analysis showed that the majority of pathways was affected or inhibited (FIG. 1A), including many growth factor signalling pathways which induce cellular proliferation and tumour progression. The most enriched canonical pathway, the tumour suppressive PTEN pathway, was indicated to be activated (z-score of 2.309). Differentially expressed genes in this pathway include RPS6KB2, KRAS, PDGFRB, SOS2, TGFBR3, CASP9, INPPL1, PIK3R1, PTK2, CBL, PDPK1, CCND1, BCAR1, and MAGI3 (FIG. 2).

[0300] Other identified pathways were significantly inhibited, including Neuregulin signalling (z-score of −2.333) and HGF signalling (z-score of −3.162). Genes from our differential expression dataset that participate in these pathways are shown in FIG. 1b and include PI3KR1, KRAS, SOS2 and PTK2. Many are important components of growth factor signalling and mitogen-activated protein kinase (MAPK) pathways, inducing nuclear signals for cellular proliferation and tumour progression.

[0301] Subsequently, IPA software was used to predict downstream effects of the observed gene expression changes on biological functions and disease processes. Out of the 100 most significant biological functions that were changed by 193a at 24 h, those that were inhibited (z-score <−2) were related to cell survival, proliferation, migration, or cancer, and those that were activated (z-score >2) were related to (tumour) cell death (FIG. 3). Furthermore, the majority of the affected biological functions (55% at 24 h) belonged to the category Cancer (Table 5, which shows categories of top 100 biological functions ranked based on the number of miRNA-193a-regulated functions (all P<0.00001) at 24 h).

TABLE-US-00005 TABLE 5 Biological function categories. 24 h Category # functions Cancer 55 Cell movement 10 Cell death and survival 9 Cell growth and development 7 Organismal development and survival 6 Nervous system function 5 Cardiovascular 3 Cell maintenance 3 DNA replication 1 Hematological disease 1

Example 2—RNA-Sequencing, Differential Gene Expression, and Pathway Analysis after Treatment of Different Cancer Cell Lines with miRNA-193a

[0302] miRNA-193a was tested in different cancer cell lines (see Table 2.1). The cells were treated with miRNA-193a as described for example 1 at different concentrations (1, 3, 10 nM). Controls (mock, untreated, and scrambled) were measured for all cell types. Assays were performed after 24 h, 48 h and 72 h. Table 2.1 shows results at 10 nM concentration at indicated time points. The results were quantified and normalized to the mock control. 10 nM was a suitable concentration, because the cells showed no signs of a toxic effect at that concentration.

TABLE-US-00006 TABLE 2.1 effect of miRN-193a on various tambours Cell cycle Cancer Cell Viability Apoptosis arrest Motility type line (96 h) (48/72 h) (72 h) (18 to 24 h) Liver HEP3B <50% <2x G2/M >50% HUH7 <50% <2x — n.a. Lung A549 <50% >2x SubG1 >50% H460 <50% >2x SubG1 n.a

[0303] miRNA-193a treatment in the cancer cell lines decreased cell viability over time as measured by either an MTS assay or by cell count. Apoptosis induction was enhanced over time as measured by a caspase 3/7 apoptosis assay. Cell cycle arrest profiles were measured performing either nuclei imaging or flow cytometry. miRNA-193a treatment induced either a G2/M or a SubG1 cell cycle arrest profile in a manner depending on the cell line. While in HUH7 no obvious cell cycle arrest profile was observed following the indicated methods, an increased apoptosis was observed indicated by Caspase 3/7 activation and enhanced cleaved-parp protein on western blot (data not shown) following miRNA-193a treatment in this cell line. This result indicates that miRNA-193a treatment affects the viability of the cells. Cell motility of two cell lines was significantly decreased after treatment as assessed via a Boyden chamber assay.

Conclusion

[0304] miRNA-193a treatment decreased cell viability partly by inducing apoptosis and by an increase in the cell cycle arrest profile. miRNA-193a treatment also decreases cell motility of cancer cells, indicating its role in the inhibition of cancer cell migration.

Example 3— Further Study of the PTEN Pathway Activation

[0305] Example 1 shows that the IPA analysis identified the tumor suppressive PTEN pathway as the most enriched canonical pathway which was activated by miRNA-193a. Here regulation of selected miRNA-193a targets is analysed at the protein level by western blotting, including: FAK (PTK2), P70S6 (RPS6KB2), PI3KR1, TGFBRIII and other important signaling molecules including P-AKT, AKT, p-ERK1/2, ERK1/2, p-c-RAF and c-RAF, all factors in the PTEN pathway.

Materials and Methods

Cell Preparation

[0306] Human cancer cell lines were cultured in appropriate media (see table below) and seeded into δ-well plates before transfection with 10 nM miRNA-193a-3p mimic as described in example 1, 10 nM scrambled random control, or mock using Lipofectamine RNAiMAX (Thermofisher). Media was aspirated 72 h after transfection and plates were stored at −80° C.

3. 1. Cell Lines Details

[0307]

TABLE-US-00007 Cell line Cancer type Medium A549 Lung (NSCLC) F-12K + 10% FBS + P/S BT549 Breast (TNBC) RPMI-1640 + 10% FBS + P/S + 0.023 IU/mL insulin H460 Lung (NSCLC) RPMI-1640 + 10% FBS + P/S HEP3B Liver (HCC) EMEM + 10% FBS + P/S HUH7 Liver (HCC) DMEM low glucose + 10% FBS + P/S + L-glutamine PANC-1 Pancreas DMEM + 10% FBS + P/S SNU449 Liver (HCC) RPMI-1640 + 10% FBS + P/S FBS: fetal bovine serum, P/S: penicillin streptomycin

Protein Isolation and Quantification

[0308] RIPA buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 0.5 mM EDTA), supplemented with protease and phosphatase inhibitor cocktails, was added to harvested cells. Lysates were centrifugated at 15,000 g for 1 h at 4° C. and clarified by removing the cell debris pellet. Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher).

Electrophoresis and Immunoblotting

[0309] Samples were separated at 120 V by SDS-PAGE on Mini-PROTEAN TGX Stain-Free precast gels (Bio-Rad). Proteins were transferred at 200 mA for 2 h to PVDF membranes in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol). The membranes were blocked using 5% milk or 5% BSA in Tris-buffered saline with Tween (20 mM Tris pH 7.6, 137 mM NaCl, 0.1% Tween). Blots were probed with primary and horseradish peroxidase-conjugated secondary antibodies. Proteins were detected using ECL reagents. Membranes were stripped in stripping buffer (62.5 mM Tris pH 6.8, 2% SDS, 100 mM 2-mercaptoethanol) for 30 min at 50° C. and reprobed as appropriate.

Results

[0310] Lysates from cells transfected with 10 nM scrambled control or 10 nM miRNA-193a-3p mimic as described in example 1 were immunoblotted to assess the protein level of selected predicted miR-193a-3p target genes as well as phosphorylation status of key signalling proteins in the PTEN pathway. In all tested cell lines (A549, HUH7, SNU449, BT549, H460, A2058, HEP3B and PANC-1) downregulation of FAK, also called PTK2, was observed in the miRNA-193a sample compared to mock and scrambled control (FIG. 4). TGFBRIII was also downregulated by miRNA-193a in cell lines where a constitutive expression level could be observed (A549, HUH7, SNU449, BT549 and H460). Protein level of PIK3R1, the regulatory subunit of PI3K, was decreased in all cell lines except SNU449. P70S6, also called RPS6KB2, was downregulated in H460, A2058 and HEP3B. Vinculin and tubulin were used as loading controls. In A549 and H460, tubulin was affected by miRNA-193a, whereas vinculin was stable, indicating that miRNA-193a does not reduce general protein levels. Additionally, we observed downregulation of AKT phosphorylation by miRNA-193a in most cell lines (A549, A2058, SNU449, HUH7, H460 and HEP3B (FIG. 5). Interestingly, miRNA-193a increased phosphorylation of ERK in at least two cell lines (A549 and A2058). In SNU449, both ERK phosphorylation and ERK total protein level were upregulated. Phosphorylation of c-RAF was only downregulated in PANC-1.

Conclusion

[0311] These results are in line with the RNA-sequencing data obtained previously. miRNA-193a-3p mimic miRNA-193a decreased protein expression of FAK, P70S6K, PIK3R1 and TGFBRIII in multiple human tumor cell lines. In addition, treatment of cells with miRNA-193a-3p mimic miRNA-193a lead to reduced phosphorylation of AKT, which could be due to downregulation of upstream signaling proteins such as PIK3R1 and FAK. Furthermore, we observed increased phosphorylation of ERK, which could be a consequence of decreased AKT activity via effects on RAF, although phosphorylation of c-RAF was decreased in only one cell line (PANC-1). Increased phosphorylation of ERK may also be the result of other upstream events, including decreased phosphatase activity or increased activity of upstream kinases.

Example 4— miRNA-193a is an Immunogenic Cell Death (ICD) Inducer

[0312] Introduction: The concept of Immunogenic Cell Death (ICD) has been defined as a unique class of regulated cell death capable of eliciting antigen-specific adaptive immune responses through the emission of a spatiotemporally defined set of danger signals known as damage associated molecular patterns (DAMPs) (Krysko et al., Nat. Rev. Cancer, 2012; Casares et al., J. Exp. Med., 2005; Kroemer et al., Annu. Rev. Immuna, 2013). The most notable DAMPs are: release of HGMB1, release of ATP and surface expression of calreticulin (CRT), as a sign of ER stress. Induction of ICD by some (specific) anticancer agents upon induction of cancer cell death leads to release of DAMPs into the tumor microenvironment (TME), which operate on receptors expressed by dendritic cells (DCs) to accelerate their maturation, and in turn stimulate presentation of tumor-associated antigens to T cells, leading to T cell activation and proliferation eventually culminating in enhanced cytotoxicity against the tumor cells, and formation of an immunological memory against the tumor antigens.

Materials and Methods

[0313] Transfection: A2058 melanoma, HEP3B and Huh7 hepatocyte, and HCT116 colon tumor cells were transfected with different concentrations of miRNA-193a as described in example 1, or a mock (“fake transfection”) control. In brief, 5×10.sup.5 A2058 or HEP3B cells were seeded in 1.5 mL complete media in δ-well cell culture plates. Both cell lines were transfected 4 h later. A 500 μL transfection mix containing 7.5 μL Lipofectamine RNAiMAX (Thermo Fisher) and the appropriate concentration miRNA-193a-3p was added to each well. Transfection conditions included were 0.1, 1, 3 or 10 nM miRNA-193a and the mock-transfected negative control. Huh7 and HCT116 cell lines were transfected 24 h later. First the media was replaced by 1.5 mL of fresh media. Then, a 500 μL transfection mix containing 7.5 μL Lipofectamine RNAiMAX and the appropriate concentration of miRNA-193a was added to each well. Transfection conditions included were 1 or 10 nM miRNA-193a and the mock-transfected negative control. All cell lines were passaged into 24-well plates 16 h after transfection by aspirating and retaining media in 5-mL tubes, washing 1× with TrypLE (Gibco), and incubating for 10 to 12 min until detached. Cells were collected with 1 mL fresh media and added to the retained media. Tubes were centrifuged for 5 min at 1,500 RPM and supernatant removed. Cells were resuspended in 500 μL fresh media and counted using a 1:1 dilution with trypan blue using the Luna-II cell counter (Westburg). 5×10.sup.4 cells in 1 mL fresh media were seeded per well.

[0314] Flow cytometry: For flow cytometric analysis at mentioned time post transfection, cells were harvested after washing 1× with TrypLE (Gibco), and incubating for 10 to 12 min until detached. For each condition, 200 μL of single cell suspensions containing 5×10.sup.4 cells were prepared in 4-mL polypropylene tubes. Cells were stained with fluorescently labeled antibodies in a 1:200 dilution. The expression of CRT was measured using a DyLight™ 488 conjugate anti-human Calreticulin (CRT) antibody (Clone FMC 75, Enzo Life science). To detect the maturation state of DCs, the surface expression of CD80, and MHC class II molecules were measured using PerCP/Cyanine5.5 anti-human CD80 antibody (Clone 2D10, BioLegend), and APC anti-human HLA-DR, DP, DQ antibody (Clone Tü39, BioLegend), respectively. Also, DAPI (BioLegend) was added at a final concentration of 2 μM, to detect live/dead cells, and dead cells were excluded from further analyses. Flow cytometry was performed using a FACSCanto II cytometer (BD Biosciences), data was analyzed with FlowJo software (Tree Star inc.).

[0315] Co-culture with CFSE labeled PBMCs: PBMCs were isolated from fresh blood buffy coat (Sanquin), using SepMate™-50 tubes (STEMCELL), following manufacturer's protocol. Ficoll® Paque Plus (SigmaAldrich) was used as the density gradient medium. PBMCs were then labeled with CFSE using CFSE Cell Division Tracker Kit (BioLegend), following the manufacturer's protocol. A2058 cells were transfected and 16 h after transfection, cells were passaged to a 24 well plate as explained before. 3×10.sup.4 A2058 cells were seeded in 0.5 mL of fresh medium into each well. Also, 0.5 mL of CFSE labeled PBMC suspensions containing 1.2×10.sup.5 PBMCs was added into each well. Same amount of PBMCs, without any A2058 cells, was cultured as “PBMC only” control condition. The co-culture was incubated at 37° C. for mentioned time. For detection of T cells, cells were stained with Brilliant Violet 510™ anti-human CD3 Antibody (Clone UCHT1, BioLegend) in a 1:200 dilution.

[0316] Isolation of monocyte from PBMC: Monocytes were isolated from PBMCs using MACS cell-separation system and pan monocyte isolation kit (Miltenyi Biotec), according to the manufacturer's protocol. In brief, PBMCs were labeled with a cocktail of biotin-conjugated monoclonal antibodies against antigens expressed on all different main cell types present among the PBMCs, except monocytes. Labeled cells then passed through columns containing micro beads conjugated to monoclonal anti-biotin antibodies. This approach enabled isolation of highly pure unlabeled and intact monocytes, by depletion of the magnetically labeled non-monocyte cells.

[0317] Co-culture of A2058 cells with monocytes: A2058 cells were transfected with 1 or 10 nM concentrations of miRNA-193a, or a mock-transfected negative control, as explained earlier. Cells were harvested 16 h after transfection, as explained before, and seeded into 96-well plates. 4×10.sup.3 cells in 100 μL fresh media were seeded per well. 4 hours later, 100 μL of DC generation medium (PromoCell), containing 4×10.sup.3-, 4×10.sup.4-, or no-monocytes (depending on the “monocyte:tumor cell ratio” condition) was added into each well. 4 days after starting of co-culture, the medium in all the wells were replaced with 150 μL of fresh DC generation medium. To have a positive control for mature DCs, 6 day after putting cells into the co-culture, component B of DC generation cytokine pack (C-28050, PromoCell) containing TNFα as a DC-maturation stimulator, was added to the wells with positive control condition (1.5 μL/well of component B cytokine pack, to have a final TNFα concentration of 5000 U/mL). On day 7 post co-culture, cells were stained with fluorescent antibodies against cell surface markers of mature DC, and analyzed with flow cytometry, as explained earlier.

Results

[0318] To investigate the effect of miRNA-193a on tumor cells, the expression of CRT on the surface of miRNA-193a transfected tumor cells was assessed by flow cytometry. As shown in FIG. 6, miRNA-193a induced expression of the CRT marker on the cell surface in A2058 cells (up to 46% after 72 h) (FIGS. 6-A and C), and in HCT116 cells (up to 65% after 96 h) (FIGS. 6-E and G), compared with mock transfected cells containing only 5% and 10% surface-CRT′ cells, respectively. The induction of CRT marker was also observed to a lesser extent in Hep3B cells (up to 8% after 72 h) (FIGS. 6-B and D), and in Huh7 cells (up to 35% after 96 h) (FIGS. 6-F and H), as compared to mock transfected cells containing only 4% and 23% surface-CRT′ cells, respectively. Moreover, via targeting two major ectonucleotidases CD39 and CD73, miRNA-193a can prevent the conversion of extracellular ATP to ADP, AMP and adenosine, and thereby retains the ATP content of the TME.

[0319] Next, we investigated whether the cytotoxic effect of miRNA-193a on A2058 cells, can stimulate the maturation process of monocytes towards full mature DCs. To address this, monocytes were put in co-culture with miRNA-193a- or mock-transfected A2058 cells. 7 days post co-culture, the frequency of cells expressing mature DC surface-markers (CD80 and MHC II) were measured with flow cytometric analysis. To make a positive control, TNFα was used as a known DC maturation inducer. As shown in FIG. 7, when monocytes were co-cultured with 10 nM miRNA-193a transfected A2058 cells in a 10 to 1 ratio, the frequency of CD80.sup.+ and MHC II.sup.+ cells were significantly increased (10× and 4.9×, respectively), compared to co-culture with mock transfected cells. These results indicate that miRNA-193a-transfected A2058 cells are able to markedly stimulate maturation of dendritic cells from monocytes in cell-based assays.

[0320] Finally, we addressed the effect of miRNA-193a on proliferation of T cells in co-culture with miRNA-193a transfected tumor cells. PBMCs were labeled with CFSE, a fluorescent non-toxic marker that can be retained within the cells and gets diluted with each cell division. Levels of CFSE measured by flow cytometry were compared between three conditions: 1) PBMCs in culture alone, 2) PBMCs in culture with mock transfected A2058 cells, and 3) PBMCs in culture with miRNA-193a 1 nM transfected A2058 cells. The results show that keeping PBMCs in co-culture with miRNA-193a-transfected A2058 cells enhanced the proliferation of T cells (FIG. 8).

[0321] Furthermore, miRNA-193a increased the vulnerability of tumor cells to PBMC-mediated cytotoxicity, as showed by fixation, staining and colorimetric quantification of survived tumor cells following co-culture with PBMCs. Interestingly, in vivo experiments in a syngeneic murine 4T1 orthotopic breast cancer model confirmed the formation of a long-term T cell mediated anti-tumor immunity in miRNA-193a treated animals, or in naïve mice that had received an adoptive T cell transfer from miRNA-193a treated mice.

[0322] Taken together, these results strongly suggest that miRNA-193a is a bona fide ICD inducer which kills the tumor cells in a way that not only stimulates PBMC-mediated cytotoxicity to enhance overall anti-tumor efficacy, but also stimulates DC maturation and activates the formation of an adaptive anti-tumor immunity.

Example 5 Effect of miRNA-193a on Human PBMC-Mediated Tumor Cell Killing Following Transfection in Human Tumor Cells

[0323] One of the most recent developments in the understanding of cancer biology is the field of immuno-oncology (10). Often tumors present the ability to evade cancer immunosurveillance, which represents one of the hallmarks of cancer (Hanahan et al., 2011) Accordingly, the main goals of cancer immunotherapy are to strengthen the patient's immune response to the tumor by improving its capacity for tumor recognition and the disruption of immunosuppressive mechanisms (Chen et al., 2017). As part of the induction mechanisms supporting pronounced immune suppression of the tumors, adenosine levels in the tumor microenvironment (TME) have recently attracted significant attention to develop novel therapeutic intervention in oncology. Adenosine in the tumor microenvironment (TME) is generated mainly by ectonucleotidases CD39 (ENTPD1; which converts extracellular adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and then to adenosine monophosphate (AMP)) and CD73 (NT5E; which is responsible for the generation of adenosine from AMP) (Stagg et al., 2010). NT5E can act as inhibitory immune checkpoint molecule, since free adenosine generated by NT5E inhibits cellular immune responses, thereby promoting immune escape of tumor cells. Indeed, adenosine is a potent immunosuppressive metabolite that is generated in response to pro-inflammatory stimuli, such as cellular stress initiated by hypoxia or ischemia. Landmark studies by Ohta and colleagues have highlighted the importance of adenosine for tumor immune escape (Ohta et al., 2006). Extracellular adenosine concentrations in solid tumors are reported to be higher than under normal physiological conditions (Blay et al., 1997).

[0324] Our transcriptome analysis identified a pool of immune related genes among the genes whose expression was affected by a mimic of miR-193a-3p as described in example 1. Among them were modifiers of TME, such as CD73. Moreover, our in vivo studies in murine models strongly suggested that miR-193a-3p, on top of its other effects, modifies the interaction between tumor cells and the immune system in a way that immune cells become more active in killing tumor cells. To assess the 10 related effect of miR-193a-3p in human cells, and also to investigate the mechanism of the miR-193a-3p mediated 10 effect, we established an in vitro assay in which, tumor cells were co-cultured together with human peripheral blood mononuclear cells (hPBMCs) isolated from healthy donor's peripheral blood, and the cytotoxic effect of hPBMCs on tumor cells was assessed with or without transfection with miR-193a-3p (see example 4).

[0325] As a first step and to establish the technical validity of such a cell-based assay, human anti CD3/CD28 T cell activator antibodies (positive control) was added to the tumor cells and PBMCs co-culture. The used activator comprises a soluble tetrameric antibody complex that binds CD3 and CD28 immune cell surface ligands. This binding results in cross-linking of CD3 and CD28, thereby providing the required primary and co-stimulatory signals for an effective T cell activation (Riddell et al., 1990; Bashour et al., 2014). As illustrated in FIG. 9, although unstimulated human PBMCs showed limited effect on tumor cell survival (co-culture), addition of anti CD3/CD28 antibodies in the co-culture led to a pronounced decrease in tumor cell survival, most likely consequent to an efficient T cell activation and subsequent T cell-mediated tumor cell killing. Interestingly, in similar study performed with primary human dermal fibroblasts, no effect of anti CD3/CD28 on fibroblast viability was observed (data not shown), strongly suggesting that experimental T cell activation does not lead to T cell-mediated normal fibroblast killing.

[0326] Next, human melanoma A2058 and NSCLC A549 tumor cells were transfected with increasing concentrations of miR-193a-3p after which they were co-cultured with human PBMCs (at different PBMCs:Tumor cells ratio) for different times. Human PBMCs from independent donors were able to induce time-dependent marked tumor cell killing upon transfection of tumor cells with miRNA-193a as described in example 1, but not the (negative) miRNA control (scramble), validating sequence-specificity of miRNA-193a activity (FIG. 10).

[0327] Taken together, our results demonstrate that transfection of tumor cells with miR-193a-3p clearly increases the vulnerability of tumor cells (e.g., A2058 and A549 tumor cells) to human PBMC cytotoxicity, by sensitizing tumor cells to PBMCs, and/or by releasing signals from transfected tumor cells to activate T cell-containing PBMCs.