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Cutting an NKG2D Ligand Short: Cellular Processing of the Peculiar Human NKG2D Ligand ULBP4

Stress-induced cell surface expression of MHC class I-related glycoproteins of the MIC and ULBP families allows for immune recognition of dangerous “self cells” by human cytotoxic lymphocytes via the NKG2D receptor. With two MIC molecules (MICA and MICB) and six ULBP molecules (ULBP1–6), there are a total of eight human NKG2D ligands (NKG2DL). Since the discovery of the NKG2D–NKG2DL system, the cause for both redundancy and diversity of NKG2DL has been a major and ongoing matter of debate. NKG2DL diversity has been attributed, among others, to the selective pressure by viral immunoevasins, to diverse regulation of expression, to differential tissue expression as well as to variations in receptor interactions. Here, we critically review the current state of knowledge on the poorly studied human NKG2DL ULBP4. Summarizing available facts and previous studies, we picture ULBP4 as a peculiar ULBP family member distinct from other ULBP family members by various aspects. In addition, we provide novel experimental evidence suggesting that cellular processing gives rise to mature ULBP4 glycoproteins different to previous reports. Finally, we report on the proteolytic release of soluble ULBP4 and discuss these results in the light of known mechanisms for generation of soluble NKG2DL.

ULBP4 is encoded at the centromeric end of the ULBP gene cluster on the long arm of human chromosome 6 by the RAET1E locus (11,17). ULBP4 glycoproteins have first been described in 2003 by Cosman and colleagues (18) as well as by Coukos and colleagues (19). Both groups identified ULBP4 based on in silico screens of human genomic sequences searching for relatives of the ULBP family members ULBP1, ULBP2, and ULBP3, which had previously been discovered during a search for binding partners of the HCMV glycoprotein UL16 and been named accordingly (14). Of note, ULBP4, like ULBP3, is not bound by the HCMV glycoprotein UL16 (18,20,21) and therefore can be considered a misnomer. Both original studies (18,19) described ULBP4 cDNA encoding for a polypeptide of 263 amino acids (including the signal peptide) and giving rise to a mature cell surface-bound protein of 235 amino acids (~27 kDa). This ULBP4 polypeptide is encoded by four exons with exon 1 encoding for the signal peptide, exon 2 for the α1 domain, exon 3 for the α2 domain, and exon 4 for the short serine-rich stalk, the transmembrane region, and a short cytoplasmic domain ( Figure 1A). This originally reported ULBP4 variant has meanwhile been termed isoform 1 by the Uniprot database 1 (22). Five additional ULBP4 isoforms (isoforms 2-6) are referenced in the Uniprot database originating from alternative splicing (23,24) and will be discussed later. Sequence analyses and phylogenetic trees constructed from the comparison of ULBP α1α2 superdomains strongly suggest that ULBP4 has diverged from other primate ULBP molecules earliest and before the separation of Old and New World monkeys (25). In addition, ULBP4 is the most polymorphic member of the ULBP family of proteins (11,26,27), although there is no functional rationale for this polymorphism. There are some reports of ULBP4 being expressed by various tumors, EBV-infected B cells, and cytokine-activated NK cells that may be relevant for the NKG2D-mediated immunosurveillance and immunoregulation in these settings (28)(29)(30)(31)(32)(33). In our approach to this peculiar NKG2DL, we realized that ULBP4 glycoproteins are poorly characterized and that there exist substantial inconsistencies when comparing the literature and publicly available databases on rather basic issues such as on biochemical properties and expression by cell lines and tissues. Hence, we set out to study expression and biochemical properties of ULBP4 molecules. Fisher Scientific, Waltham, MA, USA). All media were supplemented with 10% FCS (Biochrome, Berlin, Germany), 2 mM l-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin (both from Sigma-Aldrich), and RPMI 1640 in addition with 1 mM sodium pyruvate (Thermo Fisher). 293F cells were cultured in FreeStyle™ F17 Expression Medium (Thermo Fisher) with 0.1% Pluronic F-68 (Thermo Fisher), 100 U/ml penicillin, 100 mg/ml streptomycin, and 8 mM l-glutamine. C1R stably transfected with RSV.5neo containing the cDNA MICA*07, MICA*08 (21), ULBP4 (isoform 0), and ULBP4 (isoform 1) were cultivated in presence of 1.8 mg/ml G418.

Flow cytometry
Cells were harvested, washed twice with fluorescence-activated cell sorter (FACS) buffer (PBS, 2% FCS, 2 mM EDTA, and 0.01% sodium azide) and stained with 10 µg/ml of the primary antibody for 20 min at 4°C. Then, cells were washed again with FACS buffer and stained with DAG-488 (10 µg/ml) or GAM-APC (1.25 µg/ml) for 20 min at 4°C. After additional washing, flow cytometry analyses were performed using a FACS Canto II (BD Biosciences, Heidelberg, Germany) and data analyzed using FlowJo (Tree Star, Ashland, OH, USA). Biotinylated soluble truncated standard ULBP4 (ststULBP4) was immobilized on streptavidin-coated microspheres (Bangs Laboratories, Fishers, IN, USA) by incubating 5 µg microspheres with 5 µg/ml biotinylated ststULBP4 for 15 min at 4°C. Subsequently, ststULBP4-loaded microspheres were washed twice with FACS buffer, stained with antibodies, and analyzed by flow cytometry as described earlier.
The specific fluorescence intensity was calculated by subtracting the median fluorescence intensity (MFI) of the isotype control from the MFI of the antibody of interest.

Production of sUlBP4
A cDNA encoding for the first 222 amino acids of ULBP4 (Met  ( Figure 2A) replacing the IL-2 signal sequence and the Fc-encoding region of the vector. The resulting plasmid encoding for soluble carboxyterminally truncated and tagged ULBP4 (stULBP4) was used for transient transfection of 293F cells. Supernatants were harvested 4 days later and stULBP4 affinity-purified using HisPurTM Ni-NTA spin columns (Thermo Scientific) according to the manufacturer's protocol. Eluted stULBP4 was concentrated using Amicon ® Ultra Centrifugal Filter units (Merck, Darmstadt, Germany) and further purified by size exclusion chromatography using a Hiload™ 16/60 Superdex™ 200 column (GE Healthcare, Chicago, IL, USA) on an ÄKTA purifier (GE Healthcare). stULBP4 was collected and analyzed by SDS-PAGE ( Figure 2B). For determining concentrations of sULBP4 and sMICA in culture supernatants, ststULBP4 (His 31 to Ser 222 followed by an AviTag and a hexahistidine tag) or soluble truncated standard MICA07 (ststMICA) (Glu 24 to Gln 304 followed by an AviTag and a hexahistidine tag) was produced as described above for stULBP4 in 293F cells and purified by Ni-NTA spin columns except that the vector-encoded IL-2 signal sequence was placed in front of the coding regions of mature ULBP4 and MICA glycoproteins. Biotinylation of ststULBP4 was done using BirA Biotin-protein ligase (Avidity, Aurora, CO, USA) according to the manufacturer's protocol.

Detection of sUlBP4 and sMica
For detection of sULBP4 and sMICA, anti-ULBP4 pAb (R&D Systems) or mAb AMO1 were immobilized on MagPlex-C microspheres (Luminex, Austin, TX, USA), respectively, according to the manufacturer's instructions. Cell culture supernatants were diluted 1:1 in PBS with 1% BSA (dilution buffer) and added together with SA-PE and either biotinylated DUMO1 (for sULBP4 detection) or biotinylated BAMO3 (for sMICA detection) to 500 microspheres. Standard curves were generated by titrating ststULBP4 or ststMICA. Samples were incubated overnight at room temperature, washed twice in dilution buffer, and measured on a Luminex 100 system (Luminex). All samples were measured in triplicates.

in-solution Digestion
Purified stULBP4 protein samples were precipitated with acetone, and subsequently in-solution digestion was performed as described (36). In brief, protein pellets were washed and resuspended in denaturation buffer containing 6 M urea and 2 M thiourea. Proteins were reduced with 1 mM DTT, alkylated with 5.5 mM iodoacetamide and digested with the endopeptidase LysC (Wako) for 3 h and sequencing-grade trypsin (Promega) overnight. Peptide mixtures were concentrated and desalted using the Stop and Go Extraction technique (37).
liquid chromatography and Mass spectrometry A binary buffer system consisting of buffer A (0.1% formic acid) and buffer B (80% acetonitrile, 0.1% formic acid) was used for peptide separation on an Easy-nLC 1200 (Thermo Fisher Scientific). This system was coupled via a nano electrospray ionization source to the quadrupole-based Q Exactive HF benchtop mass spectrometer (38). Peptide elution from the in-house packed 18 cm (1.9 µm C18 Beads, Dr. Maisch Germany) column was achieved by increasing the relative amount of B from 10 to 38% in a linear gradient within 23 min at a column temperature of 40°C. Followed by an increase to 100% B within 7 min and gradients were completed by a re-equilibration to 5% B.Q Exactive HF settings: MS spectra were acquired using 3E6 as an AGC target, a maximal injection time of 20 ms and a 60,000 resolution at 300 m/z. The mass spectrometer operated in a data-dependent Top15 mode with subsequent acquisition of higher-energy collisional dissociation fragmentation MS/MS spectra of the top 15 most intense peaks. Resolution for MS/MS spectra was set to 15,000 at 200 m/z, AGC target to 1E5, maximal injection time to 25 ms and the isolation window to 1.6 Th.

Mass spectrometry Data Processing and analysis
All acquired raw files were processed using MaxQuant (1.5.3.30) (39) and the implemented Andromeda search engine (40). For protein assignment, electrospray ionization-tandem mass spectrometry (ESI-MS/MS) fragmentation spectra were correlated with the Uniprot human database (v. 2017) with manually added peptide sequences of ULBP4 starting with all possible N-termini of the mature protein. Searches were performed with tryptic specifications and default settings for mass tolerances for MS and MS/MS spectra. Carbamidomethyl at cysteine residues was set as a fixed modification, while oxidation at methionine, acetylation at the N-terminus, and conversion from Asn to Asp were defined as variable modifications. The minimal peptide length was set to seven amino acids, and the false discovery rate for proteins and peptide spectrum matches to 1%. The match-between-run feature was used with a time window of 0.7 min.
statistics Statistical analyses as detailed in the figure legends were performed using Prism 7 (GraphPad, San Diego, CA, USA).

resUlTs aminoterminus of Mature UlBP4 glycoproteins is recessed
We noted conflicting predictions for the aminoterminus of mature ULBP4 glycoproteins. In the original reports, the aminoterminus of mature ULBP4 glycoproteins, as generated by cleavage of the putative aminoterminal signal peptide, has been assigned in the absence of experimental evidence to glycine 29 along the exon 1/ exon 2 boundaries (18,19) presumably based on sequence alignments with other ULBP and MHC class I-related molecules (Figures 1A,B). However, we noted that the database Uniprot (22), in contrast to the existing literature, had assigned the aminoterminus of mature ULBP4 molecules to histidine 31 based on "manual assertion according to rules" (see text footnote 1). To clarify these conflicting predictions, we addressed this issue experimentally: soluble, carboxyterminally truncated and tagged ULBP4 ectodomains encompassing the α1, the α2 domain, and most of the serine-rich region, preceded by the "natural" ULBP4 signal peptide (stULBP4; Met 1 through Ser 222) were ectopically expressed in 293F cells, purified from the supernatants by Nickel-NTA spin columns (Figures 2A,B), fragmented by digestion with trypsin and LysC, and resulting stULBP4 fragments were subjected to liquid chromatographymass spectrometry (LC-MS). Mass spectrometry identified all expected tryptic fragments almost completely covering the mature stULBP4 polypeptide (data not shown). The aminoterminus of mature ULBP4 molecules, however, is generated by cleavage of the signal peptide during cotranslational translocation into the ER, and, accordingly, potentially resulting in the peptides GGHSLCFNFTIK, GHSLCFNFTIK, or HSLCFNFTIK, respectively, depending on the actual cleavage site. While the fragment HSLCFNFTIK was detected at high abundance, the peptides GGHSLCFNFTIK and GHSLCFNFTIK were not or only very rarely detected ( Figure 2C). These data strongly suggest that mature ULBP4 glycoproteins start with histidine 31 in line with the prediction by Uniprot and not as previously assumed with glycine 29. It remains to be determined how such a recessed β1 strand of the ULBP4 α1 domain may impact ULBP4 folding and function differently as compared with other ULBP molecules. Our mass spectrometric analyses also showed that the three putative N-glycosylation sites predicted by Uniprot at Asn 36 (α1 domain), Asn 154 (α2 domain), and Asn 212 (serine-rich region) all were glycosylated. In addition, we also detected N-linked glycosylation at Asn 82 (Figures 1B,C). Position 82 is polymorphic and occupied either by asparagine (stULBP4) or tyrosine (Uniprot reference sequence at http://www.uniprot.org/uniprot/Q8TD07), and therefore ULBP4 glycosylation can be expected to be variable within the human population with potential consequences for expression and detection. While Asn 82, Asn 154, and Asn 212 were glycosylated in almost all (>99%) tryptic fragments analyzed, glycosylation efficiency at Asn 36 appeared slightly reduced with ~90% of the fragments glycosylated (data not shown).

carboxyterminus of UlBP4 is Variable due to alternative splicing
Original studies of ULBP4 reported that exon 4, in addition to the serine-rich stalk region, entirely encodes for both transmembrane and cytoplasmic ULBP4 residues (18,19) (Figure 1). In the course of our studies on ULBP4, we characterized a commercially available EST clone (clone 601078687F1) derived from a cervical carcinoma cell line encoding for a ULBP4 variant with a divergent carboxyterminus (GenBank accession no. MH020173). This ULBP4 variant differs from the originally published sequence by an alternate and shorter cytoplasmic tail which is created by alternative splicing due to an alternative splice donor site within exon 4 (Figure 1). Accordingly, the 5′ portion of exon 4 is merged in frame with an additional exon located ~4 kb downstream (exon 5) which encodes the four carboxyterminal amino acids followed by a long 3′ UTR (Figure 1). Consequently, such ULBP4 isoforms have a distinct cytoplasmic domain shortened by eight amino acids as compared with the originally reported ULBP4 isoform 1 (Figure 1) (18,19). In addition, Uniprot lists five other ULBP4 isoforms (isoforms 2-6) based on reports of alternatively spliced transcripts for which experimental evidence of protein expression is lacking or scarce (see text footnote 1). Among these, isoform 3 is the only isoform that also includes exon 5 as reported here for the new ULBP4 isoform. Isoform 3 was identified in the course of a broad screen by the secreted protein discovery initiative (24) that used computational and experimental approaches to identify human cDNA clones encoding for putatively secreted proteins preceded by a signal peptide. Of note, the cDNA of isoform 3 is composed of exons 1, 2, 3, and 5, but lacks exon 4 which encodes for the transmembrane region. Hence, isoform 3 corresponds to an alternatively spliced and potentially soluble isoform of the new full-length ULBP4 isoform reported here to which we refer as isoform 0 in the following (Figure 1). The original studies have shown that ULBP4 isoform 1 can be expressed at the cell surface of transfected cell lines and is functionally recognized by NK cells (18,19). To assess whether the newly reported isoform 0 likewise gives rise to a ULBP4 glycoprotein that can be functionally expressed at the cell surface and consequently be recognized via NKG2D by NK cells and T cells, we generated a ULBP4-specific mAb.

ULBP4 Antibodies
The ULBP4-specific mAb DUMO1 was generated by immunizing mice with mouse mastocytoma cells P815 stably transfected with ULBP4. DUMO1 binds to ststULBP4 immobilized on microspheres (ststULBP4_im), but not to control microspheres ( Figure 3A). DUMO1 also bound to 293F cells and C1R cells transfected with either ULBP4 isoform 0 or isoform 1, but not to the corresponding mock transfectants (Figures 3C,D). Apart from corroborating specificity of DUMO1 for cellular ULBP4, these data also demonstrate that ULBP4 isoform 0 can be broadly expressed at the cell surface. While our data also indicate that ULBP4 isoform 0 may allow for a brighter surface expression than ULBP4 isoform 1, this possibility needs to be validated by further experiments. For control, we included the few commercially available antibodies said to be ULBP4-specific. While ULBP4-specific pAb and mAb 709116 bound ststULBP4_im and ULBP4 transfectants similarly to DUMO1, we did not detect binding of mAb 6E6 neither to ststULBP_im nor to the ULBP4 transfectants by flow cytometry (Figures 3A,B), although mAb 6E6 brightly detected denatured ststULBP4 in immunoblotting (data not shown). mAb 6E6 has previously been used to demonstrate ULBP4 surface expression on EBV-infected cells and on placental exosomes (31,41) as well as on ULBP4 transfectants 2 which is puzzling in the light of our results.

ULBP4 Expression
Among human tissues, we found ULBP4 transcripts by qPCR most abundantly in tissues which are of ectodermal origin such as skin, esophagus, and cervix (data not shown). A preferential ULBP4 expression in human skin has already been reported by Cosman and colleagues (18) and a strong expression bias toward tissues of ectodermal origin is also documented by publicly available databases. 3 We wondered whether isoforms 0 and 1 may be differentially expressed in such tissues but detected both at a comparable abundance in both skin and esophagus ( Figure 4A). Assessing abundance of ULBP4 transcripts in a broad variety of human tumor cell lines, we detected ULBP4 transcripts most abundantly in the cervix carcinoma cell line HeLa, whereas ULBP4 transcripts were undetectable in liver cancer cells HepG2 or in the erythroleukemia line K562 (Figure 4B and data not shown) well in line with publicly available data sets. 4 Of note, pronounced ULBP4 surface expression has been claimed based on binding of mAb 709116 for HepG2 cells by the supplier, 5 for the erythroleukemia line K562 (42) and for cytokine-activated NK cells (32). By using DUMO1 and ULBP4-specific pAb, we were unable to detect ULBP4 surface expression on HeLa, HepG2, HCT116, and HaCat cells, respectively. By contrast, mAb 709116 bound to a substantial portion of HCT116 cells and brightly stained HepG2 cells (Figure 4D) as reported by the supplier and as previously reported for another not publicly available mAb (29), although we were unable to detect ULBP4 transcripts in HepG2 cells. Collectively, our data on mAb 6E6 and 709116 advise substantial caution when interpreting results obtained with these commercially available ULBP4 antibodies and strongly suggest to validate such data by independent methodological approaches such as by qPCR and/or immunoblotting. Our data also indicate that in contrast to other NKG2DL such as MICA, MICB, and ULBP1-3, which are frequently expressed on a broad variety of human tumor cell lines (43), ULBP4 may be not or only sparsely expressed on the surface of human tumor cell lines. In fact, immunoblotting of lysates of HeLa cells revealed that the vast majority of detectable ULBP4 glycoproteins is sensitive for digestion with endoglycosidase H, and therefore retained intracellularly in the ER or Golgi complex well in accordance with the lack of detectable cell surface expression (Figures 4C,D). Assessing expression of ULBP4 isoform 1 versus isoform 0 in more than 30 human cell lines, we found that three cell lines including HeLa cells (HeLa, LNCaP, and MG-63) and primary keratinocytes expressed both isoforms, 8 cell lines expressed only isoform 0, but none expressed only isoform 1 (data not shown). Based on these findings, one may consider the possibility that the previously reported isoform 1 rather is a byproduct of inefficient splicing of ULBP4 primary transcripts, whereas isoform 0 may represent the physiologically more relevant isoform. Future research on ULBP4 should take such considerations into account.

soluble UlBP4
Isoform 3 may represent a soluble variant of ULBP4 generated by alternative splicing of the full-length isoform 0 excluding exon 4 (Figure 1). However, evidence for isoform 3 thus far relies only on analyses of transcripts/cDNA, but not on protein data (24). Therefore, existence and secretion of sULBP4 based on isoform 3 transcripts has to await validation by experimental evidence. In addition, there are reports claiming the existence of other sULBP4 isoforms generated by alternative splicing (23,44). However, for the originally reported soluble RAET1E2 isoform (Uniprot isoform 4) (44), no corresponding transcripts could be detected in a subsequent study by the same group (23). This latter study reported three other rarely occurring ULBP4 splice variants all comprising the entire exon 4 encoding for the transmembrane domain. Accordingly, these variants (Uniprot isoforms 2, 5, and 6) reportedly are cell membrane-bound variants (23), which obviously cannot be secreted without further processing. Considering physiologic expression of ULBP4 in skin, we assessed freshly isolated human keratinocytes for ULBP4 transcripts. By pairing a primer located in exon 1 with a reverse primer in the 3′ end of exon 4 (isoform 1) or in the 5′ end of exon 5 (isoform 0), respectively, we detected predominant expression of transcripts corresponding to both full-length isoforms 0 and 1. At considerably lower frequency, we also detected transcripts corresponding to isoforms 2 and 3, but not to isoforms 4-6 (data not shown). Hence, our data indicate, that sULBP4 may be generated physiologically by alternative splicing under exclusion of exon 4 (i.e., ULBP4 isoform 3) although existence of such sULBP4 glycoproteins remains to be shown experimentally. We and others had previously reported that most human NKG2DL can be released from cells either by proteolytic shedding or by exosomal secretion (12,34,(45)(46)(47). MICA (but not MICA*08), MICB, and ULBP2 have been shown to be shed by metalloproteases such as ADAM10, ADAM17, and MMP14 (48-51) while MICA*08 and ULBP3 were shown to be preferentially released in exosomes (45,52). However, neither proteolytic shedding nor exosomal release has been reported for ULBP4 possibly due to the scarcity of bona fide ULBP4-specific antibodies.
To address release of sULBP4 from ULBP4-expressing cells, we established a sandwich assay specifically detecting sULBP4 using immobilized anti-ULBP4 pAb and biotinylated mAb DUMO1. Using this assay, sULBP4 was clearly detectable in supernatants of C1R cells stably transfected with ULBP4 isoforms 0 and 1, respectively ( Figure 5B). To assess whether metalloproteases are involved in the generation of sULBP4, we added batimastat (BB-94), a potent, broad spectrum metalloprotease inhibitor, to cultures of C1R transfectants. As expected and in line with the previous reports, addition of BB-94 to C1R-MICA*07 and C1R-MICA*08 cells inhibited release of sMICA*07, but not of sMICA*08, in a dose-dependent manner ( Figure 5A). Of note, release of sULBP4 from both C1R-ULBP4 transfectants was also inhibited in a BB-94 dose-dependent manner demonstrating that both ULBP4 isoforms 0 and 1 can give rise to sULBP4 due to the action of metalloproteases (Figures 5B,C). Finally, we assessed sULBP4 in the culture supernatants of various cell lines expressing ULBP4 transcripts ( Figure 4B). In line with the highest abundance of ULBP4 transcripts in HeLa cells, we only detected low amounts sULBP4 in the supernatants of HeLa cells ( Figure 5D), but not of HCT116, HepG2, and HaCat cells (data not shown). Release of sULBP4 by HeLa cells could only partially be blocked by addition of BB-94 ( Figure 5D) indicating that HeLa cells generate sULBP4 not only by shedding of full-length isoforms 0 and/or 1 through metalloproteases. Since we detected in HeLa cells both transcripts of ULBP4 isoform 3 (data not shown) and ULBP4 proteins of a molecular mass corresponding to isoform 3 (Figure 4C), secretion of isoform 3 may explain the BB-94 independent release of sULBP4 by HeLa cells.
cOnclUDing reMarKs ULBP4 glycoproteins are among the least characterized human NKG2D ligands. Our present study suggests that this may be explained by a highly restricted expression in tissues and by cell lines as well as by the scarcity and deficiency of commercially available antibodies. While prevalence of ULBP4 transcripts indicates a strong expression bias toward tissues and cell lines of ectodermal origin, physiologic and cellular expression of ULBP4 glycoproteins by such cells remains to be addressed by future research. We here provide evidence that the aminoterminus of mature ULBP4 molecules is recessed when compared with other ULBP molecules and that also the carboxyterminus of at least a substantial portion of ULBP4 molecules substantially differs from the originally reported sequence due to previously unrecognized alternative splicing, resulting in a shortened mature ULBP4 polypeptide which we termed isoform 0. It remains to be shown which isoform is more physiological relevant. Our results also suggest that some previous results obtained with mAb 6E6 and mAb 709116 should be considered with caution. Furthermore, we here demonstrate that ectopically expressed ULBP4 is shed from the cell surface by metalloproteases giving rise to sULBP4 in a manner similarly to many other human NKG2DL. However, in HeLa cells release of sULBP4 was only in part dependent on metalloprotease activity, and possibly a major portion of sULBP4 secreted by HeLa cells is due to the alternatively spliced isoform 3. Of note, most ULBP4 glycoproteins were intracellularly retained in HeLa cells as evident from their lack of EndoH resistance and lack of surface expression, and it remains to be determined by future studies whether this is a peculiar feature of HeLa cells or a general property of ULBP4. In addition, it will be of considerable interest to determine occurrence of sULBP4, either proteolytically shed or as secreted isoform 3, in healthy or diseased state such as in patients with skin or cervical tumors. Altogether, we propose that ULBP4 should not regarded as just another NKG2DL, but that the unique cellular expression and processing of ULBP4 together with the early evolutionary separation from other ULBP family members indicates a peculiar function within the framework of NKG2D-mediated immunosurveillance.

eThics sTaTeMenT
Study includes analyses of buffy coats obtained from the Blood Donor Service Baden-Wuerttemberg-Hessen, Frankfurt am Main, after approval by the Ethics committee of the Goethe University of Frankfurt. Study includes generation of monoclonal antibodies based on immunization of mice which was carried out in accordance with current laws for animal research and approved by the Regierungspräsidium Tübingen and Darmstadt. aUThOr cOnTriBUTiOns TZ, MH, and MW designed and performed experiments and analyzed data. AS conceptualized study, designed experiments, and wrote the manuscript with the support of TZ and MH.

acKnOWleDgMenTs
The authors thank Christina Born for excellent technical assistance.

FUnDing
This work was funded by institutional funds of the Institute for Molecular Medicine.


s2fieldsofstudy: Biology Medicine

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Drug Distribution and Basic Pharmacology of Paclitaxel/Resveratrol-Coated Balloon Catheters

Purpose To experimentally investigate a new homogenously paclitaxel/resveratrol-coated balloon catheter in terms of transport of the coating to the treated tissue and local effects including histology and functional tests. Methods Adherence of the coating to the balloon was explored by in vitro simulation of its passage to the lesion. Paclitaxel and resveratrol transfer to the vessel wall was investigated in porcine coronary and peripheral arteries. Matrix-assisted laser desorption/ionization (MALDI) was used for direct microscopic visualization of paclitaxel in arterial tissue. Inhibition of neointimal proliferation and tolerance of complete coating and resveratrol-only coating was investigated in pigs 4 weeks after treatment, and the effect of resveratrol on inflammation and healing after 3 and 7 days. Results Drug loss on the way to the lesion was < 10% of dose, while 65 ± 13% was detected at the site of balloon inflation. After treatment similar proportions of drug were detected in coronary and peripheral arteries, i.e., 7.4 ± 4.6% of dose or 125 ± 74 ng/mg tissue. MALDI showed circumferential deposition. Inhibition of neointimal proliferation by paclitaxel/resveratrol coating was significant (p = 0.001) whereas resveratrol-only coating did not inhibit neointimal proliferation. During the first week after treatment of peripheral arteries with resveratrol-only balloons, we observed nominally less inflammation and fibrin deposition along with a significant macrophage reduction and more pronounced re-endothelialization. No safety issues emerged including left ventricular ejection fraction for detection of potential distal embolization after high-dose treatment of coronary arteries. Conclusions Paclitaxel/resveratrol-coated balloons were effective and safe in animal studies. Beyond acting as excipient resveratrol may contribute to vascular healing.

Introduction
Angioplasty has become the preferred local treatment of stenosis and occlusions in many vascular territories, offering high and immediate success, low invasiveness, and moderate cost. However, after successful initial restoration of patency, long-term benefit is hampered by high rates of restenosis-most frequently occurring 3-12 months after treatment. The main underlying mechanism is excessive neointimal proliferation in response to injury [1]. While additional stenting increases the initial lumen gain and prevents recoil, continuous irritation contributes to excessive scar formation beyond the initial healing process [2].
The efficacy of drug-eluting stents (DES) was the incentive for coating paclitaxel onto angioplasty balloons [3]. Release of paclitaxel from the balloons improves persistent patency but only at higher doses than paclitaxel released from coated stents.
Small molecules added to the coating have been shown to markedly improve the efficacy of balloon coating in both animal experiments and clinical trials [3,4]. A variety of additives (e.g., iodinated contrast medium, urea, surfactants, plasticizers) are in use today most of them with no biological effect or even known to cause damage to cell membranes. Other factors effecting the effectiveness of paclitaxel beyond the dose and the additive are the balloon membrane and, the coating method. Some outcome data comparing the results of clinical trials investigating different drug-coated balloons have been presented at scientific meetings [5] but they are not consistent in terms of trial design, patient populations, and methodology. In the current study, we investigated a coating with resveratrol as a potentially beneficial antioxidant [6] admixed to paclitaxel. Resveratrol is an antioxidant with a variety of actions including inhibition of platelet activation and adhesion, inhibition of blood coagulation, protective effects on the endothelium, and enhancement of nitric oxide formation. Although selected because of its effects on the adherence and release of paclitaxel, resveratrol has unique additional effects that deserve further investigation.
A variety of basic features of DCB are not addressed or cannot be explored in clinical trials. The experiments reported in this paper fill a portion of this gap. In the preclinical studies presented here we investigated adherence of paclitaxel and resveratrol to the balloon on its in vitro simulated way to the lesion, drug and resveratrol transfer to coronary and peripheral vessels in pigs, inhibition of neointimal proliferation, and effects of high-dose paclitaxel/resveratrol on the vessel wall. The methods included direct microscopic visualization of paclitaxel in arterial tissue. Furthermore, the effects of resveratrol-only balloon coatings were investigated in the same animal model.

Experimental Strategy
Experimental models were selected according to published comparative data and investigator's experience. Studies were performed in vitro (drug loss during passage through a hemostatic valve, sheath and blood) and in pig in coronary or peripheral arteries, clinical doses and significant overdose to detect potential desired or toxic effects not apparent at lower dose. Animals were sacrificed at three different points in time: a few minutes after treatment to measure peak drug concentrations, 3 or 7 days after treatment for early inflammatory reactions and healing and after 4 weeks for neointimal proliferation or toxicity. Table 1 summarizes the experiments conducted, some of which were pilot studies in preparation for an initial clinical trial. Whenever possible, treatments within one experiment were performed in randomized order. To keep the number of experimental animals as low as possible, arteries in different vascular territories were treated in some pigs as indicated in the table.

Angioplasty Balloon Catheters
The following catheters were used in the experiments: over-the-wire(OTW) design, 0.014'' or 0.035'' wire lumen, various balloon sizes as indicated in the Results section, either uncoated or coated in an expanded state with paclitaxel at doses of 3 or 5 lg/mm 2 and resveratrol as an additive ( Fig. 1) or coated with resveratrol only, 6.1 ± 0.2 lg/mm 2 . Inhibition of neointimal proliferation was studied using bare metal stents mounted on uncoated or coated balloons to enhance proliferation. In the highdose coronary group (5 lg/mm 2 ) a second coated balloon without premounted stent was inflated in the stented vessel segment. Vessel overstretch was approximately 10-30% of the vessel diameter.
Adherence of Paclitaxel and Resveratrol to the Balloon on its in Vitro Simulated Way to the Lesion Small balloons (3.5-20 mm) were passed through a hemostatic valve (on/off closure) into a blood-filled guiding catheter (Medtronic Launcher JL 3.5 6F, length 95 cm). Larger balloons (size 5.0-40 mm) were passed through a blood-filled Terumo Destination 6F introducer sheath (45 cm in length) with a membrane valve. Balloons were advanced into a vial with stirred heparinized porcine blood at approximately 37°C, kept in blood for 1 min, placed in an empty vial, inflated, and cut off the shaft. Following ethanol ([ 96%) addition, the samples were sonicated to dissolve the drug and centrifuged.

Animal Experiments
The studies were conducted in a total of 139 arteries in 65 domestic pigs weighing 24.0-30.5 kg. All applicable institutional and/or national guidelines for the care and use of animals were followed, e.g., the Commission Directive 86/609/EEC and the German Animal Protection Act, and approved by the Animal Ethics Committee (IMTR 42502-2-916, Sachsen-Anhalt, Germany).
For details of the experiments see Kelsch et al. [7]. Briefly, anticoagulants were started 2 days before treatment and continued in animals surviving for 4 weeks until sacrifice: 75 mg clopidogrel and 100 mg acetylsalicylic acid. Long-acting verapamil was started 24 h prior to treatment to reduce vascular spasm during the procedure. The pigs were sedated before general anesthesia was induced. Access was provided through an external carotid artery. Heparin (5000 IU) and 250 mg lysine acetylsalicylate were administered intra-arterially. Vessel segments were selected in coronary or iliac/ femoral arteries. The balloons were deployed as indicated and inflated for 60 s with 8-14 atm to achieve a balloon-toartery ratio of approximately 1.2. In the coronary study, 15 pigs were treated in the left anterior descending and the left circumflex artery-5 animals each with uncoated balloons, coated balloons (3 lg paclitaxel/mm 2 ), and two coated balloons (each 5 lg/mm 2 ) per vessel segment, numbers of vessels per coated balloon and uncoated control are given in Table 3. When two balloons were deployed in the same vessel segment the first one had a premounted stent and the second one did not.
In the cardiac efficacy and tolerance study, left ventricular angiography was done before any intervention and at 28-day follow-up before coronary angiography. Offline measurement of global left ventricular function was performed using the Siemens LV-EF module (Axiom Artis dBC).
After final angiography and euthanasia the hearts were examined for gross pathological anomalies. Tissue samples were taken from the left ventricle close to the septum, the right ventricle close to the right coronary artery, and the apex, and examined for histological evidence of hemorrhage, thrombosis, fibrosis, necrosis, and scar formation, as well as, evidence for endocarditis and epicarditis. Tissue slices at a thickness of 2 lm were stained with hematoxylin/eosin and then microscopically examined.
To detect potential effects of resveratrol on healing, some animals treated with resveratrol-only coated balloons were sacrificed 3 or 7 days after the intervention.
For euthanasia 10 ml supersaturated potassium chloride (25%) was intravenously injected in deep anesthesia. Treated vessel segments were dissected and either shockfrozen in cryogel (Tissue Tek O.C.T. TM , Weckert, Kitzingen, Germany) or preserved in 4% formalin solution for histomorphometry or weighted and kept at -20°C for analysis of paclitaxel and resveratrol.
Paclitaxel was visualized with a matrix-assisted laser desorption/ionization (MALDI) [8] imaging source (MALDI LTQ XL TM , Thermo Fisher Scientific) attached to a Fourier transform orbi trap mass spectrometer (LTQ Orbitrap XL TM MS, Thermo Fisher Scientific) and equipped with a mini-nitrogen laser (LTB, Lasertechnik Berlin).

Quantitative Coronary Analysis and Histomorphometry
Briefly, the CAAS II System (Pie Medical, Netherlands) was used for quantitative vessel analysis by an experienced observer blinded to treatment groups. Stented artery segments were embedded in methyl methacrylate. Final specimens were stained with hematoxylin-eosin or Masson-Goldner. Histomorphometric measurements were taken using the ''LUCIA G'' image program. Injury scores were assigned as described by Schwartz et al. [9], and inflammation was graded using scores presented by Kornowski et al. [10]. If required scores were adjusted for assessment of non-stented segments.
Non-stented peripheral vessel segments treated with resveratrol-only or plain balloons were harvested after 3 and 7 days and embedded in paraffin. In addition to fibrin deposition [11], inflammation (Mac-2, clone M3/38, Cedarlane, Burlington, Ontario, Canada) and re-  Microscopic images were analyzed using Image J software (National Institutes of Health). Macrophages were quantified by determination of Mac-2 fluorescence intensity. The proportion of lumen circumference covered by CD31positive cells was estimated in increments of 5% to assess re-endothelialization. Values are means of two sections from three section levels each.

Unused and Used Coated Balloons
Paclitaxel and resveratrol content was determined by placing unused or used balloons in a vial and adding ethanol for extraction.

Arteries (Treated Vessels Including Adjacent Segments, in Some Peripheral Arteries Including Inflated Balloons)
For extraction, a defined volume of ethanol was added. The samples were homogenized (Precelly 24 Dual Homogenizer, PEQLAB Biotechnologie GmbH, Erlangen, Germany).

Statistical Analysis
Quantitative parameters were compared across treatment groups. Continuous variables were compared by ANOVA using the software package SPSS 15.0 for Windows (SPSS Inc., Chicago, IL, USA). Data are presented as mean ± SD.

Results
Paclitaxel/resveratrol-matrix-coated angioplasty balloon catheters (Fig. 1) are used for the first 7 investigations listed in Table 1 and, in study 6 and 7, compared to the respective uncoated balloon catheters.

Paclitaxel and Resveratrol Distribution
Paclitaxel and resveratrol distribution during and following PTA are summarized in Table 2. Advancement of the DCB through a hemostatic valve, an introducer sheath or guiding catheter and contact of the folded balloon with flowing blood were simulated in vitro; the loss of drug was 5.1 ± 2.6% of dose for 5.0 or 7.0 mm balloon diameter and 0.6 ± 6.3% for 3.5 mm diameter.
Next, total inflated balloons along with the adjacent arterial wall were dissected and analyzed. At this point, 65 ± 13% of paclitaxel and 60 ± 15% of resveratrol were found at the target site trapped between the balloon membrane and the endothelial surface of the arterial wall. The difference (35%) appears to have been lost before or during balloon inflation before the blood flow is interrupted. Following balloon deflation and retraction, residual paclitaxel on the balloons was 9.6 ± 8.7% (peripheral) and 25.4 ± 16.0% (coronary). Similar proportions were found for resveratrol. Balloons in the external iliac (or femoral) and coronary arteries transferred similar proportions of paclitaxel into the arterial wall, 7.1 ± 6.1 and 7.8 ± 3.4% of dose, resulting in concentrations of 145 ± 50 and 126 ± 64 ng/mg tissue. Contrary to the findings for paclitaxel, no or only small amounts of resveratrol were detected in tissue. In summary 5% of paclitaxel was lost at the valve and in the sheath/blood before inflation and, 30% during inflation (unfolding of the balloon before blood flow is blocked), 65% was still found after inflation of the balloon, 48% of dose is washed away during deflation and retraction of the balloon and 10% remained on used balloons. The largest proportion of the drug is lost during deflation and retraction of the balloon (48%) or is never released (10%).
Paclitaxel was identified in histological slices of arteries prepared from deep-frozen samples by matrix-assisted laser desorption/ionization. Figure 2 shows the circumferential distribution in a peripheral artery shortly after treatment with a 5.0 9 40 mm balloon.

Inhibition of Neointimal Proliferation
Two studies investigated on paclitaxel/resveratrol-matrixcoated versus uncoated balloon catheters-one in coronary and one in peripheral arteries (Tables 1 and 2). Quantitative coronary angiography indicated minimal narrowing of the paclitaxel-treated vessel segments (late lumen loss 0.26 ± 0.22 mm) at the regular dose level and almost the same (0.17 ± 24 mm) at the high dose compared to late lumen loss of 1.12 ± 0.47 mm for use of uncoated balloons (p = 0.001). This finding was confirmed by histomorphometry, showing neointimal thickness of 0.42 ± 0.13 mm, 0.37 ± 0.05 mm, and 0.81 ± 0.30 mm, respectively (each of two dose levels vs uncoated: p B 0.003).
Two peripheral arteries each were treated in 13 animals; one animal treated with uncoated balloons died from internal bleeding caused by perforation of the left iliac artery. In the peripheral arteries, the larger stents were easily visible. Therefore, the layer between the contrastenhanced lumen and the stent was assessed by quantitative angiography and reported as 'neointimal thickness' in Table 2. The difference between coated (0.30 ± 0.34 mm) and uncoated balloons (1.60 ± 1.37 mm, p = 0.004) indicates suppression of neointimal proliferation by balloon coating.

Local Effects of Resveratrol
In a similar experiment as described in the last paragraph, 8 animals were treated with high-dose resveratrol-onlycoated balloons (Table 2). Four weeks after balloon dilatation and stent implantation, results were the same as for coated and uncoated balloons in terms of late lumen loss of coronary (1.38 ± 0.38 vs. 1.07 ± 0.27 mm) or peripheral arteries (1.56 ± 1.41 vs. 1.68 ± 0.58 mm). Histomorphometry did not reveal differences to vessels treated with uncoated balloons either. Compared to treatment with uncoated balloons, the inflammation score seemed to be somewhat reduced in both vessel territories, but the difference was not statistically significant.
To further elucidate the role of resveratrol, vessels of an additional 2 animals each were harvested 3 days and 7 days after treatment with uncoated or resveratrol-onlycoated balloons. Since histological parameters were consistent at different time points investigated these data were combined for comparison of treatments (Table 3). Compared to arteries treated with uncoated balloons, histology (Figs. 3, 4) of resveratrol-treated arteries revealed a tendency toward reduced fibrin deposition and significantly reduced number of macrophages (p = 0.002) as well as enhanced re-endothelialization (p = 0.01).

Tolerance
Overall, 24 animals were treated with coronary and/or peripheral paclitaxel/resveratrol-matrix-coated balloon catheters, 5 of them with high-dose coronary balloons; in the latter group 2 vessels each were treated with two balloons in the same vessel segments. No thrombi, thrombotic occlusions, or outflow obstructions were observed. None of the DCB-treated animals died, ECG changes were of no clinical significance, only blood pressure dropped somewhat during anesthesia and following nitroglycerin injection.
In the study including the high-dose coronary group, left ventricular ejection fraction was measured before treatment and after 4 weeks. It dropped in the control group (uncoated balloons) by 6.4 ± 4.2%, and in the regular and high-dose groups by 2.5 ± 8.1 and 5.4 ± 7.0%, respectively ( Table 4). Histopathology of the hearts treated with 2 high-dose balloons in two coronary arteries each revealed some increase in very slight to moderate microscopic lesions compared to the groups treated with one uncoated or one normal-dose balloon/vessel (Table 5).
Sixteen DCB-treated animals were observed for 4 weeks. All animals gained weight and showed no signs of weakness or disease. Reangiography revealed no vascular abnormalities such as thrombotic occlusions or aneurisms in the paclitaxel-treated animals. All balloons with premounted stents to further stimulate neointimal proliferation except 2 9 5 lg/mm 2 (= 2 balloons deployed in the same vessel segment, the first one with stent, the second one without stent); negative diameter stenosis indicates preserved overstretch/enlarged vessel lumen; LLL = late lumen loss (difference between lumen diameter after treatment and 4 weeks later); p coated versus uncoated; p* refers to each of the two dose levels, **) one RCA too narrow, not reached/treated

Discussion Local Drug Delivery
A variety of studies was performed to explore relevant properties of a clinically available DCB with the additive resveratrol known for beneficial vascular and cardiovascular effects.
Inhibition of injury-induced neointimal proliferation by drugs has been shown in randomized clinical trials for oral administration [12,13]  balloons, and liquid preparations [14]. Effective therapeutic agents are paclitaxel and limus drugs such as sirolimus or everolimus. The latter are preferred on coronary stents. However, although certain dose ranges have been established for different devices and procedures, little is known about the required drug concentration in tissue and how it can be achieved. Shortly after balloon deployment, we measured mean paclitaxel concentrations of 145 lg/g in porcine iliac and 126 lg/g in coronary arteries, both distinctly above effective drug concentrations [15,16]. Far less resveratrol is transferred to the arterial wall andconsidering its lower biological effect on cell proliferation-it is not surprising that no antirestenotic effect is observed with resveratrol coating without paclitaxel.

Dose Considerations
Effective paclitaxel doses differ for different modes of administration, e.g., (a) for coronary stents in the range of 100-200 lg (Taxus TM , only 10% released), (b) ca. 110 lg if dissolved paclitaxel is infused into short separated segments of coronary arteries [14], and to (c) 500-1000 lg on drug-coated coronary balloons. Data on tissue concentrations following DES placement are scarce but concentrations seem to be\ 1 lg/g [17]. It is unknown how much of the drug from a DES stays in the tissue and for how long and which proportion is rapidly cleared by the blood stream.
The need for a higher dose on drug-coated balloons compared to stents may be explained by the limited transfer of the drug from the balloon to the vessel wall. Our results with the resveratrol-matrix-coated DCB indicate that the largest proportion of the drug reaches the target site and is pushed against the vessel wall. Because solubility is very low, diffusion of the drug into tissues has no role. Drug particles are pushed against and into the tissue. A large proportion is washed away during deflation and retraction of the balloon. The tissue concentration following DCB deployment by far surpasses the solubility of the drug in aqueous media. Water-solubility of paclitaxel is 0.30 ± 0.02 lg/ml, corresponding to 0.3 lg/1 g water [19]. We found a concentration of 145 ± 50 lg/g in coronary and 126 ± 64 lg/g in peripheral arteries (Table 3), far exceeding the solubility in tissue water. This concentration is due to solid drug reservoirs, which are the source of long-lasting low tissue concentrations and efficacy, whereas the non-dissolved solid drug is inefficacious and nontoxic.
Although capillary embolism was detected on a microscopic level no relevant downstream morphological or physiological effects were seen in the animal studies in spite of high dose which is in full agreement, e.g., with the lack of troponin increase in DCB-treated patients suffering from in-stent restenosis [18].

Resveratrol
Another subject of the current investigation is resveratrol, the additive to paclitaxel. Previous comprehensive experiments and published data [20][21][22][23][24] suggest resveratrol to be a useful coating additive. The primary purpose of the additive is to modulate adherence and release of the drug. This is fulfilled by resveratrol, since drug loss during passage of the hemostatic valve and sheath and during floating in blood was \ 10% of dose compared to [ 20% of the currently leading products in similar experiments [7] and residual paclitaxel on the balloon after PTA was approximately 10%. Diminished drug loss may be explained by the lower water-solubility of resveratrol as Table 4 Left ventricular ejection fraction (%) of 5 pigs per group before treatment and at 28-day follow-up after treatment of two coronary arteries with paclitaxel/resveratrol-coated balloons ( Balloons with premounted stents except 2 9 5 lg/mm 2 (= 2 balloons deployed in the same vessel segment, the first one with stent, the second one without stent). Delta EF is the difference from day 28 to before treatment compared to X-ray contrast agent or urea (additives to the above mentioned DCB [7]). Antioxidants in general and resveratrol in particular are known for their beneficial biological effects [24], especially for protecting endothelium [25][26][27][28][29]. Consistent with these reports are our findings on reduced inflammation, fibrin deposition, presence of macrophages, and accelerated re-endothelialization. However, neither the relevance of the pharmacological effects of resveratrol to paclitaxel-coated balloons nor the impact on clinical findings has yet been explored. A direct effect on neointimal proliferation, as observed in carotid arteries of rats after implantation of resveratrolcoated stents [27] or after exposure of femoral arteries of rabbits to resveratrol solutions [23], was not seen in porcine arteries treated with resveratrol-only-coated balloons.

Limitations
The majority of the studies was planned and performed in preparation of a first clinical trial on paclitaxel/resveratrolcoated balloon catheters. They address the safe transfer of the drug to the treatment site, tolerance and efficacy of the coating in a large animal model but they did not address detailed pharmacokinetics of paclitaxel and resveratrol nor the mechanism of action. Emphasis is on information which cannot be obtained in clinical trials, such as local pharmacokinetics and histology. As usual in preclinical studies number of large animals was limited and in this study the animals were young and healthy. Results may not reflect the findings in patients and are no substitute for randomized clinical trials in a large patient population.

Conclusions
According to the findings of in vitro and animal experiments paclitaxel/resveratrol-coated DCB carry the drug to the lesion and, during inflation, transfer a sufficient portion of the dose to the adjacent vessel wall to inhibit neointimal proliferation. A larger proportion is washed away during deflation and retraction. The coating is well tolerated. Reveratrol may have a beneficial effect on inflammation and reendothelization.
Funding Contributions of B. Braun AG, Vascular Systems, Berlin, and Excellence Initiative, Deutsche Forschungsgemeinschaft are acknowledged.

Compliance with Ethical Standards
Conflict of interest US is coinventor of a patent covering resveratrol-matrix coated DCB, all other authors declare that they have no conflict of interest.
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.


extfieldsofstudy: Chemistry Medicine

text:
miR4673 improves fitness profile of neoplastic cells by induction of autophagy

Therapeutic resistance of neoplasms is mainly attributed to gradual evolution of mutational profile1. Here, we demonstrate a microRNA-mediated mechanism that effectively improves fitness of SKBR3 mammary carcinoma cells by cytoplasmic reprogramming. The reprogramming is triggered by endogenous miR4673 transcribed from notch-1 locus. The miRNA downregulates cdk-18, a cyclin-dependent kinase that regulates M-G1 transition in cycling cells2,3. Suppression of cdk-18 triggers mitophagy and autophagy. Due to high autophagic flux, oestrogen receptor-1+/progesterone receptor+/p53+ (Esr1+/Pr+/p53+) SKBR3 cells are coerced into an Esr1−/Prlow/p53−profile. Increased mitophagy in combination with proteasomal degradation of p53 transiently arrests the cycling cells at G0 and enhances radio-resistance of the SKBR3 population. These findings highlight the impact on cancer therapy of non-encoded neoplastic resistance, arising as a consequence of miRNA-mediated autophagic reprogramming that uncouples phenotype and genotype.

Introduction
Tumour resistance attributed to clonal evolution of neoplastic cells, poses a major challenge for cancer therapy 4 . Clonality of neoplastic cells is mainly considered to reflect heterogeneity of mutational landscape 5 . In the proposed linear evolutionary model, the mutational landscape is shaped by selection for phenotypes that improve the fitness profile of neoplastic cells in a stepwise and protracted manner. Recent reports suggest, however, alternative routes to tumour resistance. One such mechanism is clonal competition that leads to oscillatory dominance of resistance subclones in response to treatment 6 . Similar reports of alternating phenotypic reversal 6 suggest a more dynamic non-linear nature of tumour resistance that is not necessarily encoded. While, upstream mediators of "non-encoded" tumour resistance remain largely unknown, improved survival of a neoplastic population is underpinned by enhanced apoptotic threshold 7,8 .
Enhanced apoptotic threshold is typically caused by mutations that impair genomic surveillance mechanisms. In particular, loss-of-function mutations of the tumour suppressor gene p53 are associated with resistance to apoptosis 9 . While p53 has a short half-life in normal cells, DNA damage, among other sources of cellular stress, can stabilise p53 10 . The stabilised p53 in turn communicates cell cycle arrest and activates apoptotic signalling 10 . However, apoptosis is avoided if p53 activates concomitant autophagy as a pro-survival mechanism 11 . The increased autophagic flux promotes rapid degradation of p53 and prevents excessive accumulation of stable p53 and subsequent apoptosis 11 . Neoplastic cells commonly utilise the latter phenomenon to bypass p53-induced apoptosis via increased autophagic flux 12 . To that end, microRNAs are emerging as key regulators of autophagy in neoplastic cells 13 . While microRNAs inhibit various stages of the autophagy cascade from induction to vesicle nucleation 14 , there is no evidence for the positive induction of autophagy. Herein, we report efficient induction of autophagy by miRNA-4673 encoded in intron 4 of human notch-1 locus. The miRNA-induced autophagic flux depletes cytoplasmic p53 and improves post-radiation survival of neoplastic cells. The observed non-encoded induction of resistance is underpinned by miRNAmediated cytoplasmic reprogramming.

Results
MiR4673 was initially detected in clinical samples of breast cancer patients 15 . Transcript level of miR4673 in neoplastic breast tissue is significantly higher than other tissues (Fig. 1a). We, therefore, selected the SKBR3 mammary carcinoma cell line with an extra copy of ligand-independent Erbb-2 (Her2) and high endogenous expression of miR4673 (Fig. 1a) to study potential involvement of miR4673 in induction/suppression of nonencoded resistance mechanisms.
MiR4673 does not target p53, catenin-β1, notch-1 and erbb-2 directly. Hence, offsetting the anti-catabolic signalling input from hyperactive Erbb2 can be accomplished by post-transcriptional (Ago2-dependent RNA silencing) and/or post-translational (mTOR-dependent autophagy) mechanisms (Fig. 1c, d bottom). As predicted, miR end+exo cells demonstrated Ago2 high /mTOR low profile in contrast to the Ago2 low /mTOR high fingerprint of AS Δzen cells. The miR-mediated Ago2 high profile communicates enhanced Ago2-dependent RNA silencing 19 . The mTOR low profile, on the other hand, is consistent with increased autophagic flux 20 . In addition to autophagy, downregulation of mTOR reduces protein synthesis via ribosomal S6 kinase 21 and 4E-BP1 (Eukaryotic Translation Initiation Factor 4E Binding Protein 1) 22 . The findings suggest that the Ago2 high /mTOR low profile uncouples genotype and phenotype by non-selective depletion of intracellular protein reservoir. We, therefore, investigated potential impact of phenotypic uncoupling on the breast cancer diagnostic fingerprint 23 informed by availability of Esr1, Pr, and Erbb2 proteins.
Phenotype/genotype uncoupling distorts the diagnostic portrait of neoplastic cells Forced signalling by the microRNA (miR end+exo ) coerced an Esr − and Pr low molecular profile in the neoplastic cells (Fig. 2a) similar to the miR-induced depletion of catenin-β1 and p53. The miRNA also restored normal membranous representation of Erbb2 in Erbb2 high neoplastic cells by overriding transcriptional input from the extra copy of the gene (Fig. 2a). In agreement with our findings, proteasomal degradation can effectively exhaust the cellular reservoir of Erbb2 24 and ESR-1 25 despite active transcription and translation. The supervisory dominance of proteasomal activity over transcription is often invoked during development to offset transcriptional noise and induce signalling robustness 26 . The same capacity is recapitulated in response to stressors where p27 induces autophagy and cell cycle arrest as a prosurvival mechanism 27 . Interestingly, we noted reemergence of nuclear Ki-67 ( Fig. 2a) following inhibition of the endogenous miRNA (AS Δzen ) that suggests entry into the interphase 28 . One interpretation is that enhanced protein degradation in miR end+exo SKBR3 cells impacts on availability of cyclins and arrests the cells at G0 phase. In a parallel scenario, serum starvation arrests cycling cells at G0 by upregulating autophagy 29 . The quiescent cells arrested at G0 are expected to become more resistant to stressors due to autophagy. As expected, the miR end+exo cells changed morphology, became more adherent and demonstrated higher survival rate and faster recovery following UVC irradiation (Fig. 2b). Enforced adherence to matrix suggested stabilised integrin-based focal adhesions. Focal adhesion kinase signalling, in turn, inhibits cyclin-dependent kinase inhibitors p21 and p27 30 and improves tolerance to DNA damage 31 caused by irradiation. We, therefore, sought a molecular mechanism that explains the increased protein degradation and G0 arrest instructed by miR4673 signalling.
A faux stress signal improves fitness of neoplastic cells by invoking a G0 molecular signature We identified Cdk-18, the closest mammalian homologue of yeast Pho85 cyclin-dependent kinase, as a potential target of miR4673 (Fig. 3a). In yeast, Pho85 acts as a nutrient sensor 32 that licences M-G1 transition in cell cycle 3 . Pho85 downregulates autophagy 33 to propel cell cycle progression after mitosis. In response to nutrient deprivation, pho85 induces arrest at G0 to improve the survival of yeast cells. Likewise, the human homologue (PCTAIRE Protein Kinase 3 or cdk-18) acts downstream of cyclin-A2 34 to drive cell cycle progression. The impact on cell cycle of Cdk-18 occurs in part by relieving Fig. 1 Uncoupling of genotype/phenotype propelled by miR4673. a Endogenous miR4673 expression in various human tissues extracted from miRmine database 78 . See methods for detailed list of tissue samples represented in dots. Gel shows endogenous expression of miR4673 in SKBR3 cells detected by stem-loop PCR method at sequential time points 10 min. apart. b Diagrammatic representation of genotype/phenotype interface in Erbb2 high SKBR3 cells. Inhibition of autophagy, microtubule destabilization, and nuclear import defects characterise the phenotype of SKBR3 cells. c Immunohistochemical panel highlighting the impact of miR4673 on bistable population-level transition of neoplastic cells. Application of the exogenous miRNA (miRend + exo) leads to repression of p53, cateninβ1 and Notch-1 and suppression of the endogenous miRNA (AS ΔZen ) reverses the observed trend. The molecular fingerprint subsequent to amplification of miR4673 strongly contrasts with the molecular signature after application of Paclitaxel (Scale bars = 40 μm). d Quantification of immuno-labelling profile from c following up-and downregulation of the endogenous miRNA activity. Dynamics of four non-encoded clonal profiles are demonstrated by the presence or absence of the first and second markers in four combinations ( + / + , −/−, + /−, −/ + ) in stacked bar plots. The −/− to + / + conversion signifies non-encoded modulation of p53/p21/notch-1 profile by the miRNA endoplasmic reticulum stress 35 and concomitant autophagy 36 to restore activity of cyclins that drive progression through G1/S 37 . Sequential application of exogenous miRNA (200 nM, 2 × 10 5 cells) resulted in near-complete post-transcriptional inhibition of cdk-18 (Fig. 3a). The inhibition of cdk-18 inactivates cofilin 34 that manifests as reduced actin depolymerisation in miR end+exo cells (Fig. 3b). The miR4673-mediated suppression of Cdk-18 also explains the upregulated autophagy/mitophagy in miR end+exo cells 35 (Fig. 3b). The diminished mitochondrial activity subsequent to mitophagy not only contributes to radio-resistance 38 (Fig. 2b) but can also indirectly trigger redox-dependent polymerisation of actin microfilaments 39 (Fig. 3b). Enhanced mitophagy downstream to miR4673 signalling effectively offsets Paclitaxelinduced perinuclear localisation of mitochondria shown to trigger apoptosis by imposing an oxidative burden on the nucleus 40 (Fig. 3b). Arguably, communication of faux nutrient deprivation signal (Cdk-18 − equivalent to Pho85 − ) by miR4673 triggers a key stress-response with concomitant autophagy/mitophagy that improves plasticity and survival capacity of neoplastic cells. The application of exogenous miRNA-4673 enhanced the induction of autophagy and progression to autophagosome formation and subsequent degradation of the cargo ( Fig. 4a-d). The inhibition of autophagic flux partially restored the molecular profile of SKBR3 cells in miR end + exo cells (Fig. 4e, f). The latter also explains entry into interphase following the suppression of miR4673 (ki-67 + AS Δzen cells in Fig. 2a) that communicates an absence of stressors. We further dissected the impact of faux metabolic stress (cdk-18 − profile) on transcriptional fingerprint of cycling miR end+exo cells.
The cateninβ1-cylinD1-retinoblastoma axis, as the major propellant of cell cycle 41,42 , was focused upon. A key transcriptional signature of miR end+exo cells was downregulation of cyclin-D1 short isoform that lacks the cyclin box required for cdk4/6-mediated phosphorylation of retinoblastoma protein 41 (Fig. 5a). A similar trend was evident in alternative splicing of catenin-β1 following the amplification of miR4673. The transcriptional suppression of truncated cyclin-D1 eliminates decoy protein (alternatively spliced with no cyclin box) that can inhibit the Rb-E2f cascade at G0 (Fig. 5a). The elimination of decoy truncated catenin-β1 (with actin binding and phosphorylation domains) enhances the phosphorylation likelihood of full length protein required for trans-signalling 43 (Fig. 5a). Transcriptional repression of Notch-1 (antagonist of catenin-β1) further energises the activated cateninβ1-cylinD1-Rb axis at M-G1 transition during cell  cycle. Activation of the latter axis in G0-arrested cdk-18 − / miR end+exo cells occurs in anticipation of progress into G1. Enhanced autophagy, by proteasomal degradation (Fig. 1c), offsets the activity of cateninβ1 − cylinD1-Rb axis and arrests the miR end+exo cells at G0. Another prominent transcriptional change following amplified miR4673 signalling was silencing of Brca2 and X-Ray Repair Cross Complementing 3 (Xrcc3). Brca2 and Xrcc3 are critical components of homologous recombination (HR) machinery 44,45 . Inhibition of BRCA2 and XRCC3, which suggests inactivation of HR, is also a signature of G0/early G1 46 . The inhibition of HR alters the balance in favour of error-prone non-homologous end-joining (NHEJ). The remarkably accelerated dynamics of NHEJ compared to HR in repairing DNA breaks (≈30 min versus > 7 h) 47 contributes to post-radiation survival and recovery 48,49 of miR end+exo SKBR3 cells. Transcriptional silencing of Brca2 may in part reflect the depletion of upstream activator ESR-1 by autophagy (Fig. 2a) 50 . On the other hand, the mitophagy-mediated switch to glycolysis exhausts the cellular supply of NAD + and decreases NAD + /NADH ratio that in turn triggers transcriptional silencing of brca-1 51 . The findings point to a stress signature invoked by endogenous miR4673 signalling compatible with G0 molecular fingerprint (Fig. 5b). The latter is aligned to the ancestral role of Pho85 (Cdk-18 homologue) in signalling G0 arrest in response to stressors 3 . Hence, suppression of cdk-18 by miR4673 communicates a faux stress signal analogous to stress-mediated inhibition of Pho85 that triggers pro-survival autophagy and stress adaptation. In addition to G0 lengthening, downregulation of brca1, brca2, and xrcc3 combined with proteasomal degradation of p53 can potentially relax the G1-S checkpoint and accelerate interphase during which cells are more susceptible to DNA damage (Fig. 5c). Acceleration of interphase by suppressing the G1 checkpoint is a feature of embryonic stem cells 52 and it is not uncommon for neoplastic cells to recapitulate aspects of ontogeny. In our proposed model for reprogramming of cell cycle by miR4673 activity, miR end+exo cells dwell longer in G0 (stress-resistant phase) and progress more rapidly through G1 (sensitive phase).
We confirmed miR4673-induced cell cycle reprogramming by dissecting the population growth dynamics of miR end+exo cells SKBR3 cells. In contrast to smooth exponential growth of control cells, sequential application of the exogenous miRNA induced periods of dormancy followed by episodic population growth (Fig. 6a). This behaviour was consistent with transient cell cycle arrest at G0 (dormancy) followed by fast synchronised cycling and division (episodic population growth) (Fig. 6b). The unchanged growth rate despite arrest at G0 indicated a reciprocal shortening of interphase (Fig. 6a). Reprogramming of cell cycle by miR4673 can potentially improve resistance profile of the neoplastic cells by lengthening G0 (resistant phase) and simultaneous shortening of interphase (sensitive phase). We next explored endogenous transcriptional oscillations of miR4673 and the associated downstream genes that instruct windows of resistance in neoplastic populations. The cells were first synchronised at G0 by serum starvation. Subsequent to addition of serum, a high-resolution temporal fingerprint of endogenous miR4673 and associated genes, that instruct the population fitness of SKBR3 cells, was generated.

Endogenous oscillations of miR4673 and the downstream cascade portray a variable resistance landscape
Transcriptional activity of the endogenous miRNA and the host gene notch-1 oscillated with a periodicity of 2 h (Fig. 7). These results are consistent with the reported periodicity of notch-1 oscillations 53 . Notably, amplitude of the miRNA oscillation ([max]:[min]≈80-fold) was much higher than notch-1 oscillation ([max]:[min]≈3-fold). In addition, notch-1 oscillation lagged behind that of the endogenous miRNA by 1 h. Concurrent oscillation of the endoplasmic reticulum stress was measured based on shift in the alternative spliced product of X-Box Binding Protein 1 (xbp-1) 54 . High endoplasmic reticulum stress (characterised by abundance of short alternative splicing product of xbp-1) temporally accorded with peak miR4673 transcriptional activity (Fig. 7a). The transcription of rad51 recombinase (a core component of HR) and PPP1R13B (ASPP1; a positive regulator of p53 activity) and xrcc3 showed a periodicity of 1-1.5 h and was repressed concurrent with the second peak activity of (see figure on previous page) Fig. 4 Enhanced autophagic flux underpins the altered molecular fingerprint of amplified miR4673 signalling in SKBR3 cells. a Live-imaging analysis of miR end and miR end+exo SKBR3 cells 24 h after transfection with RFP/GFP-tagged LC3 reporter plasmid, ptfLC3 (Scale bars = 40 μm). While miR end+exo SKBR3 cells express RFP, miR end cells express both RFP and GFP. The latter is consistent with the enhanced incorporation into the autophagosomes and fusion with lysosomes of RFP/GFP-LC3 subsequent to the amplification of endogenous miR4673. b Co-localisation analysis of RFP and GFP (based on Spearman's coefficient and overlap) confirmed uncoupled RFP and GFP signals in miR end+exo cells as a result of upregulated autophagy. c Western blot analysis of autophagy-related proteins LC3, Beclin-1, p62 (SQSTM1), and Hsp-70. The reduced level of LC3-II in miR end+exo SKBR3 cells due to autophagy was consistent with live-imaging analysis of ptfLC3-transfected cells. Likewise, enhanced levels of Beclin-1 and p62 (SQSTM1) proteins in miR end+exo SKBR3 cells indicated amplified induction/assembly phase of autophagy. Eventually, we investigated the availability of HSP-70 as a chaperone for protein folding and autophagy. HSP-70 was retrieved at a lower level from miR end+exo SKBR3 cells consistent with enhanced degradation phase of autophagy. d Semi-quantitative analysis of c based on intensity of the bands normalised to the maximum intensity (100%). e Reduction of autophagic flux by the application of Bafilomycin A1/chloroquine restored the reduced cytoplasmic pool of p53, PR, and Cateninβ1 in miR end+exo SKBR3 cells to a level comparable to that of control miRend cells (Scale bars = 100 μm). f Quantification of immuno-labelling profile from e following the reduction of autophagic flux by the application of Bafilomycin A1/chloroquine The graphs show altered molecular fingerprint of SKBR3 cells following the amplification of endogenous miR4673 signalling (* indicates p < 0.05, ** indicates p < 0.01). E1 and E2 are sequential electroporations 24 h apart. We used two separate sets of primers to fingerprint the 5ˊand 3ˊnotch-1 locus. b Schematic demonstration of system-level miR4673 interactions that improve fitness profile of neoplastic cells. Interactions triggered by the miRNA signalling eventually improve anti-oxidant defence capacity and accelerate DNA repair mechanisms through non-homologous end-joining. c Schematic representation of cell cycle shows prolonged G0 subsequent to the amplification of the endogenous miRNA activity that instructs inhibition of G1/S and G2/M cycle checkpoints simultaneously miR4673 (Fig. 7). Transcription from brca-1 was, however, totally in phase with oscillations of miR4673 and showed a periodicity of 2 h. We fingerprinted Snail family members as potent inducers of G0 arrest and neoplastic resistance 55 . The transcriptional fingerprint of twist-1, a trans-acting partner for notch-1 56 , was temporally aligned to the latter gene and showed a periodicity of 2 h (Fig. 7). The transcriptional oscillation of snai-2 as a downstream target of Notch-1 57 accorded with twist-1 expression (periodicity: 1.5 h). These oscillatory fingerprints foreshadow the alignment of endogenous miR4673 oscillations to the windows of resistance that occur at intervals of ≈2 h. We, therefore, tested the contribution of miR4673 to the adaptation of SKBR3 cells to the acute stressor, ionising radiation.

Endogenous signalling by miR4673 improves the survival likelihood of SKBR3 cells
We modelled the contribution of endogenous miR4673 to post-radiation survival of the neoplastic cells based on Poisson statistics and assumption of "single-target singlehit" 58 . The binary model assumes that a single exposure to the radiation is sensitive enough to kill the impacted cell. Hence if the radiation hits a cell "k" times, the likelihood of escape from the impact of radiation or P(k = 0) in Poisson probability space corresponds to: Where m (dose correction parameter) is proportional to the probability of exposure to the radiation. To measure endogenous variability in resistance, SKBR3 cells were exposed to ionising radiation at sequential time points 1 h apart. Notably, the post-radiation survival rate (SR) of control SKBR3 cells (with endogenous miR4673) oscillated with a periodicity of 2-3 h (Fig. 8a). The endogenous difference between the maximum SR (t 3 ) and the minimum SR (t 1 ) in the control group based on Poisson statistics Pðk ¼ 0Þ ¼ e Àm ) m t 1 =m t 3 ¼ LnðSR t 1 Þ=LnðSR t 3 Þ ¼ Lnð0:15Þ=Lnð0:47Þ ¼ 2:5 indicated ≈2.5-fold reduction of the impact of radiation on population from t 1 to t 3 (measured based on day 1 post-radiation SR). We then translated the ≈2.5-fold reduction of radiation impact at a population-level to the The growth by 1.14 unit of the parameter "m", that is given a default value of 1 based on the "one hit one target" model, corresponds to a halved probability of death by irradiation for individual cells. Therefore, two hits are required to cause lethality based on Poisson's model. On the other hand, a single exogenous dose of miR4673 (Fig. 8b, c) reduced the impact of radiation on neoplastic cells at t 4 by m t 4 2 =m t 4 ¼ LnðSR t 4 2 Þ=LnðSR t4 Þ ¼ Lnð0:18Þ=Lnð0:49Þ ¼ 2:4 where m 2 and m refer to irradiated miR endo+exo and control groups, respectively. Enhanced survival rate after exogenous application of miR4673 at t 4 (2.4-fold) approached the maximal endogenous radio-resistance . c Post-radiation survival of miR end+exo SKBR3 cells (200 nM exogenous miR4673). The surviving cells were exposed to a second dose of radiation after day 19. Note the reversal of survival rate at t 3 of control cells and t 3 , t 4 , t 6 of miR end+exo cells (dotted lines). d Post-radiation survival (day 1) of control miR end and miR end+exo SKBR3 cells (200 nM exogenous miR4673) subsequent to inhibition of autophagy by Chloroquine. Note the reduced survival rate in miR end+exo similar to that observed for miRend cells. e Schematic diagram demonstrates the contribution of miR4673 to the fitness landscape of SKBR3 cells. The amplified activity of miR4673 increases the incidence of resistant periods. The higher incidence of resistant temporal windows (bottom graph) increase the likelihood of treatment in a period where neoplastic cells are refractory (2.5-fold). Further, amplified miR4673 signalling enhanced the incidence of radio-resistant periods by two fold at 100 nM (Fig. 8b) and by three fold at 200 nM (Fig. 8c). Remarkably, a second dose of radiation at day 19 triggered a reversal of the survival profile at t 3 in the control group (dotted lines in Fig. 8a-c) and at t 4 in the miR endo+exo population consistent with the nonencoded nature of radio-resistance. The application of Chloroquine reduced the mean survival rate in both miR end and miR end+exo SKBR3 cell to ≈10% (Fig. 8d). We, therefore, propose a model for the miR473-induced enhancement of neoplastic fitness profile (Fig. 8e) where 50% increased resistance of individual neoplastic cells anticipates a 2.5-fold improvement of the population survival rate at any time point. In our proposed model, miR4673 signalling also increases the incidence of resistant temporal windows.

Discussion
The findings reported herein attest to plasticity of molecular signatures that inform non-encoded resistance in a neoplastic population. Plasticity results from miR4673 signalling that upregulates proteasomal degradation and overrides the transcriptional fingerprint of SKBR3 cells. The resultant phenotypic uncoupling significantly improves fitness profile of the neoplastic population. Phenotypic uncoupling is particularly manifest in the repressed profile of p53 following the amplification of miR4673.
Tumour suppressor p53 is a key target of human oncogenic miRNAs. MiR-125b, for example, downregulates p53 by targeting the 3ˊ-untranslated region (UTR) of the transcript in human neuroblastoma cells and human lung fibroblast cells 59 . Likewise, miR504-mediated suppression of p53 enhances the resistance of neoplastic cells by increasing the threshold of apoptosis 60 . Hybridisation to the UTR, however, triggers partial inhibition of the targeted transcript 61 . In contrast, indirect inhibition of p53 by miR4673-mediated autophagy leads to nearcomplete suppression of the protein. Direct targeting of xrcc3 by miR4673 complemented indirect suppression of p53. Notably, loss-of-function mutations of XRCC3 attenuate the apoptotic signalling in neoplastic cells 62 . At a population-level, reprogramming of cell cycle by miR4673 (transient arrest at G0 followed by accelerated interphase) maximises the resistance of neoplastic cells. Enhanced autophagy is the key to the major phenotypic manifestations of miR4673 signalling.
The autophagy-mediated nuclear uncoupling reflects a developmental capacity to reprogram the proliferating cells by depletion of cytoplasmic protein repertoire 63 . The most remarkable example of the nuclear uncoupling occurs during reprogramming of the highly differentiated oocyte into undifferentiated zygote by complete degradation of maternal proteins 64 . Mitophagy is also commonly employed in physiological processes 65 . Differentiation of the reticulocytes, for example, is governed by amplified mitophagy 66 . Recapitulation of autophagymediated reprogramming enables neoplastic cells to adapt to stressful situations 67 . In response to the application of anticancer drugs autophagy can act as a pro-survival mechanism 68 . Autophagy also improves resistance to ionising radiation 69 . The induction of autophagy by miR4673 is unique as most reported human microRNAs are negative regulators of autophagy 14 . Some other aspects of the autophagic reprograming by miR4673 are also worthy of attention.
A distinguishing feature of signalling by miR4673 is system-level interactions that complement autophagymediated reprogramming and enhance the potency of the microRNA signalling. Suppression by autophagy of p53, for example, is complemented by transcriptional inhibition of xrcc3. Another distinguishing feature of miR4673 signalling is acute endogenous oscillations of the miRNA (every 2 h). These transcriptional oscillations are aligned to temporal behaviour of notch-1, the host locus of miR4673. Transcription from Notch-1 locus and the associated downstream mediators oscillates with a periodicity of 2 h 53 . The transcriptional oscillations of miR4673 accommodate windows of neoplastic resistance with potential implication for therapy. The phenotypic variability may also impact on the accuracy of diagnosis by distorting the molecular fingerprint of tumours. Finally, the non-encoded nature of the described reprogramming mechanism suggests that the capacity may be available to other neoplastic cells with active transcription from notch-1 locus. Even in the absence of active transcription, epigenetic remodelling may unlock this capacity and alter the behaviour and molecular profile of the neoplasm acutely.
From a diagnostic perspective, cytoplasmic reprogramming by autophagy can potentially alter the molecular fingerprint of neoplastic cells. Non-encoded reprogramming of SKBR3 cells by miR4673, for example, communicates a Esr1 -/Pr low /erbb2 low subtype that can potentially indicate more aggressive treatment options. In parallel, the enhanced fitness profile not only improves real-time survival of neoplastic cells but also facilitates accumulation of iatrogenic genetic changes that may in turn propel a switch from non-encoded to encoded (genetic) resistance associated with poor prognosis 70 .
From a therapeutic perspective, non-encoded homogeneity is not without consequences. Our findings demonstrate that miR4673 improves radiation resistance of SKBR3 cells by 50% that translates into a 2.4-fold higher survival rate of the population. The improved fitness results from increased mitophagy that enhances antioxidant capacity of the neoplastic cells. The inhibition of homologous recombination machinery to empower nonhomologous end-joining (NHEJ) cascade also improves DNA repair capacity. This effect is particularly manifest in resistance to ionising radiation where accumulating evidence indicates an indispensable role of NHEJ in repair of DNA damage 48,49,71 .
In conclusion, we have uncovered a non-coded route to tumour resistance (as opposed to mutational resistance) that results from intrinsic oscillations of miR4673 expressed at high levels in breast cancer. The signalling cascade utilised by the microRNA may be targeted to reveal the true molecular fingerprint of tumour by inhibition of the autophagic reprogramming. Suppression of the endogenous miRNA may also improve the response to therapy.

Materials and reagents
All chemicals were purchased from Sigma-Aldrich Inc. unless stated otherwise. All primers were purchased from IDT DNA.
Cell culture SKBR3 cell line was purchased from ATCC cell bank (ATCC ® HTB-30™). Cells were cultured in standard McCoys-5A medium (Sigma-Aldrich ® , M4892) supplemented with 10% FBS (Gibco™) and were split at ≈75% confluence. All the experiments were performed at a similar confluency rate. Serum starvation method was utilised to synchronise the cycling cells at G0.

Electroporation
For electroporation, cells were harvested and resuspended in a modified "Intracellular buffer" 72

UVC irradiation
SkBr3 cells were were collected and transferred into a 6well-plate and cultured for 2 days. A 30 Watt UVC generator at a distance of 40 cm from the plate was used as a source of ionising radiation. The UVC lamp was turned on for 3 × 20 s with 10 s intervals. The cells were then immediately supplemented with fresh McCoys media.

RNA isolation
RNA was isolated using a Trizol reagent. Bromochloro (0.1 M) was added per 1 ml TRIzol ® Reagent (Ambion, 15596018). The tubes were vortex then centrifuged for 15 min at 12000 g at 4°C. The aqueous phase was mixed with 2 μl of 5 μg/μl linear polyacrylamide (ambion) and 500 μl of isopropanol. The solution was then vortexed then left to incubate at room temperature. The solution was then centrifuged for 1 h at 20,000 × g at 4°C. The supernatant was discarded followed by the pellet being washed with 1 ml of ice cold 200-proof ethanol. The sample was then vortexed followed by centrifugation for 30 min at 12,000 × g at 4°C and the supernatant was discarded. The RNA pellet was left to air dry, then resuspended in 50 μl of RNAse free H 2 O (Ambion™ AM9937) and stored at -80°C.

Real-time qPCR
Real-time quantitative PCR (38 cycles) was performed using SensiFAST™ SYBR ® Lo-ROX reagents (BIOLINE ® ). Reaction mix comprised of 2 μl of cDNA, 400 nM inner primers (1.5 μl/primer), 10 μl of 2x SensiFAST SYBR Lo-ROX Mix, and 5 μl of PCR-grade water on a Stratagene ® Mx3000P real-time PCR instrument. Average efficiency of PCR amplification for each gene of interest was quantified based on a linear regression model using the LineRegPCR software 73 . The relative (normalised to β-actin) expression ratio of gene of interest (test: control) was then calculated using the efficiency of amplification (Eff.) values based on the method proposed by Pffai 74 as follows: To generate the temporal fingerprints of genes, we used a variation of the above method: Melt curve analysis and visual inspection of the amplified products on 2% agarose gel electrophoresis confirmed the presence of target amplicons. Relative expression of transcripts for each gene was plotted as ratio between the average concentration of transcript in the test and control groups normalised to the average concentration of β-actin transcripts in both groups (above formula). The primers used in the current study are presented at Supplementary  Table 1.

Stem-loop PCR for detection of endogenous miRNA
Small RNA from proliferating HNPs was isolated using mirVana™ miRNA isolation Kit (Ambion) according to the manufacturer's protocol. Detection of the miRNA-4673 was accomplished using stem-loop PCR as described elsewhere 75 . Specific primers were designed for reverse transcription and stem-loop RT-PCR amplification of the miRNA as shown in Supplementary Table 2 After PCR amplification, products were run on a 1.5% agarose gel at 5 V/cm, stained in SYBR Gold for 45 min and destained in 1 × TAE buffer for 30 min before imaging. Imaging was performed using the MultiDoc-It™ Imaging System.

Immunohistochemistry
After blocking in incubation buffer containing 0.1 M PBS, 1% BSA, 0.1% Tween-20, and 5% normal goat serum (for detection with rabbit Abs) or 5% normal rabbit serum (for detection with mouse Abs) for 40 min, sections were incubated with the primary antibodies overnight at 4˚C and secondary antibodies for 1 h at room temperature as per Supplementary  76 . Two isoforms of 345 and 395 kDa have been identified 77 . In western blotting of lysates of the multiple myeloma cell line, IM-9, the antibody labels bands of 345 and 395 kDa, identical to isoforms of Ki-67 protein (manufacturer's technical information).

Autophagy inhibitors
To inhibit the autophagic flux, Bafilomycin A1 (Sigma) and Chloroquine (Sigma) were applied at a final concentration of 10 nM and 50 μM, respectively. The cells were incubated with the inhibitors for 16 h and then processed for immunohistochemical staining as outlined before.

Live-imaging analysis of pftLC3 autophagy reporter
The autophagy reporter plasmid, ptfLC3, was a gift from Tamotsu Yoshimori (Addgene plasmid #21074). SKBR3 cells were electroporated with the reporter plasmid alone or in combination with miR4673 as described before. For live-imaging analysis cells were cultured in 6-well plates overnight and transferred into the live-imaging platform (Leica DMI6000B live cell imaging microscope). Phase contrast images were captured every 30 min for 16 h from multiple wells (20x magnification). To analyse mitotic activity, images were imported into FIJI (ImageJ) platform.

Transmission electron microscopy
For TEM analysis, samples were fixed in Karnovsky's fixative overnight at room temperature followed by postfixation in OsO 4 for 1 h. Preparations were dehydrated in graded alcohols and embedded in low viscosity resin (TAAB Laboratory and Microscopy, United Kingdom). Ultrathin sections were mounted on Pioloform/formvar coated slot grids, stained in uranyl acetate and lead citrate and examined in a Phillips CM120 BioTWIN electron microscope.

Bioinformatics analysis
The expression profile of miR4673 in various human tissues was extracted from the miRmine database as per Supplementary Table 4. The raw output was analysed and a graph generated using the GraphPad software.

Quantification and statistical analysis
SPSS statistical software (SPSS v.16, Chicago, Illinois, US) was used for the statistical analysis of data. The relative expression levels of genes of interest were compared using non-parametric Mann-Whitney U-test. In the present study, a p-value < 0.01 was considered as statistically significant.
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