miR-184 represses β-catenin and behaves as a skin tumor suppressor

doi:10.21203/rs.3.rs-3216354/v1

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miR-184 represses β-catenin and behaves as a skin tumor suppressor

https://doi.org/10.21203/rs.3.rs-3216354/v1

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published 26 Feb, 2024

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miR-184-knockout mice display perturbed epidermal stem cell differentiation. However, the potential role of miR-184 in skin pathology is unclear. Here, we report that miR-184 controls epidermal stem cell dynamics and that miR-184 ablation enhances skin carcinogenesis in mice. In agreement, repression of miR-184 in human squamous cell carcinoma (SCC) enhances neoplastic hallmarks of human SCC cells in vitro and tumor development in vivo. Characterization of miR-184-regulatory network, suggests that miR-184 inhibits pro-oncogenic pathways, cell proliferation, and epithelial to mesenchymal transformation. Of note, depletion of miR-184 enhances the levels of β-catenin under homeostasis and following experimental skin carcinogenesis. Finally, the repression of β-catenin by miR-184, inhibits the neoplastic phenotype of SCC cells. Taken together, miR-184 behaves as an epidermal tumor suppressor, and may provide a potentially useful target for skin SCC therapy.

Squamous cell carcinoma (SCC) is the second most common form of non-melanoma skin cancer. The causes for SCC are environmental including exposure to ultraviolet radiation, chemical exposures, viral infection such as human papillomavirus, or host factors such as genetic susceptibilities, skin tone and immunosuppression (Silpa & V, 2013). SCC may develop from actinic keratoses, which are precursor lesions that may progress to SCC. Prevention by treatment of actinic keratoses or early detection and surgical excision of SCC allows clinical cure (Eduardo Calonje et al., 2020). However, tumors that evolve, become infiltrative and aggressive, may recur locally or develop metastasis. Treatments to locally advanced or metastatic SCC are sparse, and include radiation therapy, chemotherapy, EGFR inhibitors and recently immunotherapy (Fania et al., 2021; Kitamura et al., 2020). Response to these therapies is only partial, and there are no good histological markers that can predict therapy response. Therefore, there is a need to better understand the molecular pathways that control cancer progression and cancer stem cells. This understanding will promote the development of better diagnostic and prognostic markers and efficient therapeutic measures (Smirnov et al., n.d.).

One of the principal pathways that are hyper-activated and pro-tumorigenic in SCC is the Wnt/β-catenin pathway (Sherwood & Leigh, 2016). Wnt signaling controls several signal transduction pathways and is implicated in the development/progression of multiple cancers when aberrantly regulated. Recent evidence has highlighted a potentially critical role for Wnt signaling in both the development and progression of cutaneous SCC (Sherwood and Leigh 2016). The level of β-catenin, a major intracellular signal transducer of the Wnt pathway, was increased in cutaneous SCC samples compared to normal skin, as demonstrated by immunohistochemistry (Lan et al., 2014). In addition, local activation of Wnt/β-catenin was observed in cutaneous SCC tumors but not in healthy skin (Sobel et al., 2015). The identification of Wnt pathway as upregulated and as pro-tumorigenic in cutaneous SCC, suggests that targeting Wnt compounds may represent a pertinent therapeutic strategy (Sherwood and Leigh 2016).

Micro ribonucleic acids (miRNAs) are small non-coding RNAs that function as post-transcriptional repressors. miRNAs typically bind to complementarity sequences of the 3′-untranslated region of messenger RNA of their target genes, thereby, inducing mRNA degradation or repressing mRNA translation (Bartel, 2004). Aberrant expression, amplification or mutations in miRNA coding genes have been associated with the silencing of a tumor suppressor target gene or activation of oncogenes, leading to initiation, progression and drug resistance of different human malignancies (Akçakaya et al., 2011)(Calin & Croce, 2006a)(Khan et al., 2019) (Calin & Croce, 2006a; Si et al., 2019). Nevertheless, very little is known about the role of miRNAs in modulating cutaneous SCC. The unique properties of miRNAs makes them an optional therapeutic target by antisense oligonucleotides, an approach that is under investigation in pre-clinical studies (García-Sancha et al., 2019).

miR-184 is a highly evolutionary miRNA conserved from fly to human. Four different point mutations in different sites of pre-miR-184 were associated with multiple and severe eye abnormalities that lead to blindness (Hughes et al., 2011a). Previous reports, suggested that miR-184 may act as a tumor suppressor or oncogenic miRNA (Wong et al., 2008)(Chen et al., 2018) (Cheng et al., 2015) (Malzkorn et al., 2010) (Malzkorn et al., 2010) (Foley et al., 2010) (Wong et al., 2009), suggesting a context-dependent role for miR-184 in cancer. Ablation of miR-184 in mice resulted in augmented epidermal stem cell proliferation and skin hyperplasia (Nagosa et al., 2017a), suggesting that miR-184 may play a role in skin cancer.

Here, we report that miR-184 acts as a skin tumor suppressor gene in mouse and human models. miR-184 is down-regulated in chemically induced tumorigenesis and its repression in human cutaneous SCC enhances neoplastic cell hallmarks. We characterized miR-184-regulatory network and propose that by inhibiting canonical Wnt/β-catenin, miR-184 represses SCC phenotype.

Cell culture

C12C20 human SCC cells were grown in G medium (containing 60% Dulbecco’s modified Eagle’s medium (DMEM, Gibco), 30% DMEM/F-12 (Gibco), 10% FCII serum (Hyclone), 5µg/ml insulin (Merck), 0.5µg/ml hydrocortisone (Merck), 10ng/ml EGF (Peprotech), 0.2mM adenine (Merck) and 1% penicillin/streptomycin). Lentiviral vector of control orAnti-miR184 sequences were generated and lentivirus production was done as described (Altshuler et al., 2018). For in vitro assay, cells were plated at 0.5 × 10⁶ cells per well (six-well plate) and 24 hours later, infected with lentivirus in the presence of Polybrene (10 mg/ml) (Sigma-Aldrich). After 48 hours, green fluorescent protein (GFP) expression was validated to assure efficient infection, followed by sorting of GFP-positive cells.

In vitro assays

For colony formation assay, cells were seeded at 500 cells/well of 6-well culture plates in duplicates. For β-catenin inhibitor experiments, 24 hours after seeding, cells were treated with PKF118-316 (0.1 µg/ml, Sigma) and 9 days later, the colonies were fixed with 4% formaldehyde, stained with 0.05% crystal violet (Sigma), counted by examination of five microscopic fields and quantified by Image J. For trans-well migration assay, we used modified Boyden chambers with a polycarbonate Nucleopore membrane (Corning, Corning, NY). Filters (6.5 mm in diameter, 8µm pore size) were coated with fibronectin 10µg/ml (biological industries, Israel). Cells were maintained in serum-free media for 10 hours prior to migration assay. Cells (2x10⁵) were then suspended in 200 µl of serum-free medium, seeded in duplicates on the upper part of each chamber, and the lower compartment was filled with 600µl medium with 10% FCS. After overnight incubation at 37°C in a 5% CO₂ incubator, non-migrating cells on the upper surface of the filter were wiped with a cotton swab and migrated cells on the lower surface of the filter were fixed with 4% formaldehyde, stained with 0.05% crystal violet (Sigma), and counted by examination of five microscopic fields. The percentage of surface covered by migrated cells was calculated using Image-Pro-Premier (9.3.2).

Animal models

All experiments were performed according to ethical committee approval (IL-127-07-17). The miR-184-knockout strain (C57BL/6N) and genotyping are detailed in (Nagosa et al., 2017). For two-stage skin carcinogenesis, the dorsal hair of 6–7 weeks old females of each genotype (6 biological replicates) was removed using hair shaver. One week later (7–8 weeks of age, the resting hair growth phase) 7,12-dimethylbenz[a]-anthracene (DMBA; Sigma; 50 mg/0.1 mL acetone) was topically applied to the shaved area. After one additional week, 12-O-tetradecanoylphorbol-13-acetate (TPA; Sigma; 10 µg/0.1 mL acetone) was topically applied twice weekly for 20 weeks. For xenograft model, NOD/SCID immunodeficient mice (BALB/c background) were purchased (Envigo RMS, Israel). SCC stably infected (10⁶) C12C20 cells in phosphate-buffered saline (PBS) were subcutaneously injected into the flank of females (five biological replicates). Tumor volume was measured twice a week using a caliber and analyzed at endpoint. For staining, fresh tissues were fixed (4% PFA in PBS) for 12 hours at 4°C, then dehydrated and stained as described (Nagosa et al., 2017a)(Bhattacharya et al., 2019).

RNA extraction, real-time polymerase chain reactions and sequencing

Cells were washed with PBS, lysed using TRI-Reagent (Sigma) and RNA was extracted according to the manufacturer’s instructions. RNA from mouse epidermis was extracted with RNeasy Micro Kit (Qiagen) according to manufacturer recommendations. cDNA was prepared from RNA as previously described (Nasser et al., 2018). Primers that were used for quantitative polymerase chain reaction (qPCR) were ITGFβ4 forward 5'-CTCTCCATCGGCAGCCAG-3' and reverse 5'-CACCAGCAGTCAGGCGAGAG − 3', SOX2 forward 5'-TACAGCATGTCCTACTCGCAG and reverse 5'- TAGGAAGAGGTAACCACAGGG-3', K15 forward 5'-GACGGAGATCACAGACCTGAG-3' and reverse 5'-CTCCAGCCGTGTCTTTATGTC-3', P63 forward 5'-GTCATTTGATTCGAGTAGAGGGG-3' and reverse 5'-CTGGGGTGGCTCATAAGGT-3'.

RNA sequencing was performed at the G-INCPM center (https://g-incpm.weizmann.ac.il/). The epidermis of newborn mice was immersed with 70% ethanol for 2 min, twice, the skin was dissected and incubated on a culture plate (the dermis side down) with Dispase (65mg in 50 ml PBS and 1:500 penicillin/streptomycin (Gibco, Life Technologies) at 4⁰C ON. Next, the epidermis was gently separated from the dermis and RNA was extracted using RNeasy Micro Kit (Qiagen) according to manufacturer instructions. The RNA quality was tested using 'Qubit' and only samples that passed quality parameters (OD260/280 ≥ 1.8, OD260/230 ≥ 2, RIN number ≥ 8) were included. Sequencing libraries were prepared using mRNAseq. SR60 reads were sequenced on 2 lane(s) of an Illumina HiSeq2500v4. The sequencing yield was ~ 29 million reads per sample. A single KO sample that displayed low quality control and poor sequencing outcome was removed from the analysis. Bioinformatics: Poly-A/T stretches and Illumina adapters were trimmed from the reads using cut adapt; resulting reads shorter than 30bp were discarded. Reads were mapped to the M. musculus GRCm38 reference genome using STAR, supplied with gene annotations downloaded from Ensembl (and with End-to-end option and out FilterMismatchNoverLmax was set to 0.04). Expression levels for each gene were quantified using htseq-count, using the gtf above. Differentially expressed genes were identified using DESeq2 with the betaPrior, cooksCutoff and independent Filtering parameters set to False. Raw P values were adjusted for multiple testing using the procedure of Benjamini and Hochberg. Pathway analysis, gene ontology analysis in which the sets of differentially expressed genes enriched were analyzed using Humanmine (https://www.humanmine.org//humanmine/portal.do).

Western Blot analysis, immunofluorescence and Immunohistochemistry

Cells were washed with cold PBS and lysed in RIPA lysis buffer (Tris-HCl 10mM, 10mg/ml Deoxycholate, 1% NP40, 1% SDS, 150mM NaCl, protease inhibitors cocktail (Roche)) on ice, transferred to a new Eppendorf and then incubated for 5 min at 95⁰. Next, cells were sonicated for 20 seconds at Amp 20%. The protein concentration was measured by DC Protein kit (Bio-Rad). Proteins (40µg) were separated on an 8% polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS) and transferred to nitrocellulose membranes (Bio-Rad). The membranes were blocked with 5% milk (Bio-Rad) and probed with one of the following antibodies diluted in blocking solution: rabbit α-K15 (1:1000 Santa Cruz), rabbit α-NICD (1:1000 Cell signaling), mouse α-P63 (1:1000 Santa Cruz), rabbit α-GAPDH (1:1000 Cell signaling), rabbit α-β-catenin (1:1000 Sigma) at 4°C over night, then washed three times, exposed to secondary antibody (1:5000) (1hour at room temperature) and washed three times before revealed using ECL kit (Biological Industries).

For immunofluorescent staining, secondary antibodies were AlexaFluor 488 and 593 nm (Renium). Immunohistochemistry was performed as previously described (Avitan-hersh et al., 2020). Briefly, paraffin blocks were sectioned at 5µm and stained with indicated antibodies. Slides were subjected to antigen retrieval using Dako Target Retrieval Solution and incubated for 1 hour with blocking solution (Dako). The blocked sections were incubated overnight at 4°C with the indicated antibodies and diluted 1:200 in Dako Antibody Diluent. Slides were then washed twice with PBS, incubated for 2 hours at 250C with relevant secondary antibody diluted 1:200 in Dako Antibody Diluent, washed again with PBS, revealed with Simple Stain AEC solution (Histofine), and counterstained with hematoxylin. Images were taken by Nikon Eclipse NI-E upright microscope and Zeiss LSM880 confocal microscope. For quantification, the indicated number of fields of at least 3 different biological replicates were imaged and the indicated mean fluorescence intensity was calculated by ImageJ software. For lineage tracing experiments, colonies were imaged (focus on basal layer), and ImageJ software allowed colony identification and size analysis, using 5 fields of 3 biological replicates.

Statistical analysis

The error bars in all the measurements represent the means +/- standard deviation (SD). Student’s t-test was performed to determine the respective statistical significance. Differences were considered statistically significant from a p‐value below 0.05.

miR-184-ablation leads to hyper stem cell activation and susceptibility to cancer

miR-184 induces a commitment switch to epidermal differentiation (Nagosa et al., 2017a) and miR-184-deficient animals display epidermis hyperplasia (Fig. 1A, S1A). We therefore hypothesized that miR-184-knockouts may suffer from imbalanced stem cell/tissue dynamics that may lead to higher susceptibility to cancer. To explore that, we first established double transgenic UBC-Cre^ERT2; R26R-Brainbow^2.1 mice on the background of Mir184 wild type (WT) or knockout (KO). In this multi-color “Confetti” lineage tracing system, transient exposure of adult (~ 2-months old) transgenic animals to tamoxifen (3–4 days), permits the stochastic and irreversible labeling of epidermal cells with one out of four fluorescent protein-coding genes (i.e. cytoplasmic red (RFP) or yellow (YFP), membrane cyan (CFP) or nuclear green (GFP)) (Amitai-Lange et al, 2015; Nasser et al, 2018; Altshuler et al, 2021) (Fig. 1B-C). Mice were sacrificed at different time points post induction and tail epidermal tissues were imaged by whole mount confocal microscopy. Efficient and comparable induction of Confetti reporters in the basal epidermal layer was evident in both genotypes (Fig. 1D, upper pictures, T0). Interestingly, the average clonal size (i.e. the average number of basal cells in a clone) was significantly higher in KO animals, suggesting that miR-184 represses stem cell proliferation and epidermal tissue turnover. The hyper stem cell activity in KO mice implies that miR-184 may act as a suppressor of skin cancer development. To test that, we explored whether miR-184 expression influences the development of tumors in the two-stage carcinogenesis model. Adult mice of each genotype were treated with a single dose of the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) followed by exposure (twice a week) to the tumor promoter proliferation/inflammation inducer (TPA) for five months (Fig. 2A). Eleven weeks post-DMBA application, macroscopically tumors appeared in the back of KO animals, while in the WT counterparts tumors firstly appeared ~ 14 weeks post-induction (Fig. 2B). Of note, by week 20, miR-184-KO mice developed a significantly higher number of tumors (Fig. 2B) that were also larger in size (Fig. 2C-D), compared to those formed in WT mice. Hematoxylin and eosin (H&E) staining have shown that, as expected, most tumors displayed features of papilloma and that in WT animals, tumors were hallmarked by keratin pearls and cellular atypia (Fig. S1B-C). Interestingly, the tumors of miR-184-deficient mice were larger, and included infundibular cyst structures that penetrated the deep dermis. This indicate that loss of miR-184 potentiates tumor formation in the murine epidermis. To further corroborate this hypothesis, we explored whether the levels of miR-184 are changed during cell transformation in WT mice. RNA was extracted from normal skin (healthy) or from tumors (DMBA/TPA induced) of WT mice. TaqMan-real time PCR analysis of miR-184 revealed that the levels of miR-184 were significantly (~ 60%) lower in the tumors as compared to the healthy tissue (Fig. 2E). Altogether, these experiments strongly suggest that miR-184 serves as epidermal tumor suppressor in mice.

miR-184 inhibits neoplastic phenotype of human cutaneous squamous cell carcinoma

To explore the function of miR-184 in human cell transformation, we first validated the expression of miR-184 in human skin by in situ hybridization on paraffin sections. In line with its expression in mouse, miR-184 is undetectable in the dermis and in basal epidermal cells, whereas it is readily detected by early committed cells at the spinous layer (Fig. 3A). TaqMan real-time PCR analysis confirmed that the levels of miR-184 are significantly lower in C12C20 SCC cells compared to healthy foreskin derived epidermal cells (Fig. 3B). While this is in agreement with the observed reduction of miR-184 expression in DMBA/TPA tumor formation, it can be due to different genetic background. To address the role of miR-184 in the same cells in a controlled experiment, we stably infected the human SCC C12C20 cells with a pLKO-H2B-GFP lenti-viral vector that contains an antagonizing anti-miR-184 (AM184) sequence and an empty pLKO-H2B-GFP vector was used as control (Ctl) (Fig. S2A). The efficient infection of AM184 or Ctl vectors was evident by the expression of green fluorescent protein (GFP, Fig. S2B) that facilitated cell purification by cell sorter (Fig. S2C). Repression of miR-184 was validated by real-time PCR (Fig. S2D). Next, SCC C12C20 cells stably expressing the Ctl vector (C12C20/Ctl) or the AM antagonist (C12C20/AM) were subjected to colony formation assay. As shown in Fig. 3C-E, a higher number of colonies that were also larger in size was detected in C12C20/AM184 cells as compared to the control counterparts. Next, we evaluated the ability of SCC cells to migrate through polycarbonate membrane in a Boyden chamber assay. As shown in Fig. 3F-G, the migration potential of AM-infected cells was much higher, compared to the control C12C20 SCC cells. These results suggest that miR-184 inhibits C12C20 SCC cell transformation in vitro and may play a role as tumor suppressor in vivo. To further test the anti-tumorigenic role of miR-184 in human C12C20 SCC cells in vivo, we applied xenograft model. C12C20 SCC cells stably expressing AM184 or Ctl vector were subcutaneously injected to the flank of immune-deficient (NOD-SCID) mice and tumor growth was followed over time. Interestingly, AM184 expressing human C12C20 SCC cells generated a significantly larger tumors (Fig. 3H), strongly suggesting that miR-184 acts as a tumor suppressor in human skin SCC.

miR-184-repression of Wnt/β-catenin contributes to SCC phenotype

To unravel the molecular network that is regulated by miR-184, we performed bulk RNA sequencing (RNAseq) of the newborn epidermis of miR-184 WT and KO mice. The epidermis of each genotype was isolated as detailed in Methods, RNA was extracted and samples that passed the quality test were subjected to RNA sequencing. The expected sample segregation was according to the genotype and 216 genes were found to be differentially expressed between WT and KO groups (fold change > 2, adjusted P value > 0.05, > 30 reads) (Fig. S3A). To unveil the signaling network of miR-184, we preformed comparative analyses of datasets based on gene lists using Metascape algorithm (https://metascape.org/gp/index.html) (Fig. 4A-B). Interestingly, genes that are involved in key processes of epidermal terminal differentiation (e.g. keratinization and protein crosslinking), were significantly down-regulated in KO mice (Fig. 4B), suggesting that miR-184 is essential not only for early epidermal differentiation (Nagosa et al., 2017a), but also for the broader processes of terminal differentiation and cornification. Among the upregulated genes, were also genes that are involved in hair keratinization (Fig. S3B). Importantly, a significant enrichment of genes that play a role in pro-tumorigenic pathways such as mesenchyme morphogenesis, epithelial to mesenchymal transition and proliferation were upregulated in miR-184-KO mice (Fig. 3B). Selected upregulated and downregulated genes were confirmed by real time PCR (Fig. 4C). Canonical Wnt pathway also increased in miR-184 KO epidermis, in line with elevated levels of β-catenin protein in miR-184 deficient cells (Fig. 4D).

Previous studies highlighted the role of Wnt/β-catenin signaling in the development of cutaneous SCC (Sherwood and Leigh 2016). To test whether there is a link between miR-184-depletion and high β-Catenin levels in cancer, we performed Western blot analysis on lysates of control and miR-184-depleted human and murine tumor cells. Interestingly, the levels of β-catenin were consistently higher in miR-184 deficient murine tumors (Fig. 5A). Similarly, the levels of β-catenin were significantly higher in human C12C20/AM184 SCC cells grown in vitro or in xenografts (Fig. 5B-D), suggesting that miR-184 repression of β-catenin is conserved between mouse and human. Since WNT/β-catenin pathway plays a pro-oncogenic role in human SCC, we next explored whether the tumor suppressor function of miR-184 is mediated by repression of repression of Wnt/β-Catenin pathway. To test that, we performed rescue experiments using the β-Catenin/TCF4 inhibitor, PKF118-310. As shown, the β-catenin/TCF4 inhibitor PKF118-310 significantly attenuated the colony formation capacity of C12C20 SCC cells. By contrary, the AM184 enhanced colony formation, and the PKF118-310 inhibitor reduced the number of colonies in C12C20/AM184 SCC cells (Fig. 4F-G). The same trend was also observed in A431 SCC cell line, suggesting that this axis is broad. Here too, AM184 enhances the colony formation of A431 SCC cells whereas PFK118-310 reverts this effect (Fig. 5F-G). Collectively, these data suggests that the tumor suppressor function of miR-184 is, at least partly, mediated by the repression of WNT/β-catenin.

Previous studies have shown that aberrant expression of miRNAs, often due to genetic or epigenetic modifications, has critical influences on the initiation and progression of human malignancies (Calin and Croce, 2006), or cancer stem cell activity (Khan et al. 2019). The present study indicates that miR-184 behaves as a tumor suppressor in the skin. This conclusion is based on: (i) augmented formation of tumors in the 2-stage carcinogenesis model in miR-184-deficient mice, (ii) enhanced colony formation and cell migration in miR-184-knocked down SCC cell lines, (iii) inhibitory effect of miR-184 on human SCC tumor growth in vivo.

miRNAs are considered as fine-tuning molecules that are essential for the gene expression network. Typically, the depletion of a single miRNA would lead to mild defects with no major abnormality unless stress was applied (Park et al., 2010). However, few miRNA encoding genes were associated with human genetic diseases. miR-184 belongs to this group of essential miRNAs as point mutations in miR-184 were linked with abnormal eye development and blindness (Hughes et al., 2011) and its ablation in mice leads to epidermal hyperplasia (Nagosa et al., 2017). Identification of the role of such key miRNAs in human diseases and developing novel treatments based on miRNA targeting is a new and expanding field of study. Such miRNA-based drugs are being tested in clinical trials for targeting oncogenes and reinstating the expression of tumor suppressor genes.

While the importance of these essential miRNAs is clear, their function in vivo, and their mechanism of action in skin SCC are poorly defined. In this study, transcriptome analysis of the epidermis of miR-184-KO mice revealed that several pro-oncogenic pathways, including Wnt/β-catenin, epithelial-to-mesenchymal cell transformation and cell proliferation were upregulated. Wnt/β-catenin pathway plays an important role in SCC, however, the upstream regulators of this pathway in skin SCC are not well defined. The present study suggests that miR-184 plays a role in the repression of this axis and that downregulation of miR-184 facilitates the enhanced activity of β-catenin which in turn increases the susceptibility to development and progression of SCC. This suggests that a miR-184 mimic compound may target Wnt/β-catenin, and can represent a pertinent therapeutic strategy for skin SCC (Sherwood and Leigh 2016). Interestingly, this study suggests that the expression levels of miR-184 expression are reduced in skin carcinogenesis. This suggests the oncogenic pathways act upstream to repress the transcription and/or stability of miR-184.

It was previously demonstrated that miR-184 represses epithelial stem cell proliferation and induces differentiation via Notch-induced epidermal differentiation (Nagosa et.al. 2017). The transcriptome analysis in the present study further confirms this hypothesis and suggests a role for miR-184 in repression of stemness and induction of differentiation. Mechanistically, Wnt/β-catenin repression by miR-184 can be explained by activation of Notch, since genetic ablation of Notch1 significantly induced elevated activation of Wnt/β-catenin (Nicolas et al., 2003). Thus, this accumulating evidence suggests that miR-184 may be regulating SCC cancer stem cells. The molecular signature of cancer stem cells is strikingly different from that of their healthy stem cell counterparts (Yang et al. 2015) and they possess a potent capacity for self-renewal and differentiation, which is essential for tumor growth (Miao et al. 2019). Therefore, the prediction from our study would be that a miR-184 mimic compound would allow cancer stem cell differentiation, potentially via Notch activation and/or Wnt/β-catenin repression to inhibit SCC tumor growth.

Finally, a better understanding of the function of key miRNAs in vivo and specifically, in pathological conditions has therapeutic implications. In the case ofmiR-184, point mutations in a single allele led to a complex eye dystrophy (Hughes et al., 2011). Future studies should examine the influence of miR-184 mutations on Wnt/β-catenin pathway in miR-184-related eye dystrophy, and the impact of miR-184 mutation on SCC development and progression in these patients. The newly developed technologies to deliver miRNA mimics or anti-miRNA antagonists provides an opportunity for the development of efficient cancer therapy as well as restoring normal function in miR-184 deficient patients.

Conflict of Interest Statement – the authors declare no conflicts of interest

Author Contribution Statement – RSF and EAH performed study concept and designed experiments; LT, GK, GY, SN, SB and IB performed experiments. LT, GK, GY and IB provided acquisition, analysis and interpretation of data, and statistical analysis; NI, IV, IB provided technical and material support. LT, DA, RSF and EAH performed development of methodology and wrote, reviewed and revised the manuscript; All authors read and approved the final paper.

Funding Statement – EAH received funding from Israel Cancer Association (20210135, 20221521). R.S.F received funding from Horizon Europe - Innovative Europe 802601, Israel Science Foundation (1308/19 and 2830/20) and the Rappaport Family foundation (035-10).

Ethics Statement- All experiments were performed in accordance with the Declaration of Helsinki, ethical committee approval no. IL-127-07-17.

Akçakaya, P., Ekelund, S., Kolosenko, I., Caramuta, S., Özata, D., Xie, H., Lindforss, U., Olivecrona, H., & Lui, W. O. (2011). miR-185 and miR-133b deregulation is associated with overall survival and metastasis in colorectal cancer. International Journal of Oncology, 39(2), 311–318. https://doi.org/10.3892/ijo.2011.1043
Altshuler, A., Verbuk, M., Bhattacharya, S., Abramovich, I., Haklai, R., Hanna, J. H., Kloog, Y., Gottlieb, E., & Shalom-Feuerstein, R. (2018). RAS Regulates the Transition from Naive to Primed Pluripotent Stem Cells. Stem Cell Reports, 10(3), 1088–1101. https://doi.org/10.1016/j.stemcr.2018.01.004
Avitan-Hersh, E., Feng, Y., Oknin Vaisman, A., Abu Ahmad, Y., Zohar, Y., Zhang, T., Lee, J. S., Lazar, I., Sheikh Khalil, S., Feiler, Y., Kluger, H., Kahana, C., Brown, K., Ruppin, E., Ronai, Z. A., & Orian, A. (2020). Regulation of eIF2α by RNF4 Promotes Melanoma Tumorigenesis and Therapy Resistance. Journal of Investigative Dermatology, 140(12), 2466–2477. https://doi.org/10.1016/j.jid.2020.04.008
Bartel, D. P. (2004). MicroRNAs: Genomics, Biogenesis, Mechanism, and Function. Cell, 116(2), 281–297. https://doi.org/10.1016/S0092-8674(04)00045-5
Bhattacharya, S., Serror, L., Nir, E., Dhiraj, D., Altshuler, A., Khreish, M., Tiosano, B., Hasson, P., Panman, L., Luxenburg, C., Aberdam, D., & Shalom-Feuerstein, R. (2019). SOX2 Regulates P63 and Stem/Progenitor Cell State in the Corneal Epithelium. Stem Cells, 37(3), 417–429. https://doi.org/10.1002/stem.2959
Calin, G. A., & Croce, C. M. (2006a). MicroRNA signatures in human cancers. Nature Reviews Cancer, 6(11), 857–866. https://doi.org/10.1038/nrc1997
Calin, G. A., & Croce, C. M. (2006b). MicroRNA signatures in human cancers. In Nature Reviews Cancer (Vol. 6, Issue 11, pp. 857–866). https://doi.org/10.1038/nrc1997
Chen, D., Li, J., Li, S., Han, P., Li, N., Wang, Y. I., & Du, S. (2018). Mir-184 promotes cell proliferation in tongue squamous cell carcinoma by targeting SOX7. Oncology Letters, 16(2), 2221–2228. https://doi.org/10.3892/ol.2018.8906
Cheng, Z., Wang, H. Z., Li, X., Wu, Z., Han, Y., Li, Y., Chen, G., & Xie, X. (2015). MicroRNA-184 inhibits cell proliferation and invasion , and specifically targets TNFAIP2 in Glioma. 1–13. https://doi.org/10.1186/s13046-015-0142-9
Fania, L., Didona, D., Di Pietro, F. R., Verkhovskaia, S., Morese, R., Paolino, G., Donati, M., Ricci, F., Coco, V., Ricci, F., Candi, E., Abeni, D., & Dellambra, E. (2021). Cutaneous squamous cell carcinoma: From pathophysiology to novel therapeutic approaches. Biomedicines, 9(2), 1–33. https://doi.org/10.3390/biomedicines9020171
García-Sancha, N., Corchado-Cobos, R., Pérez-Losada, J., & Cañueto, J. (2019). MicroRNA dysregulation in cutaneous squamous cell carcinoma. International Journal of Molecular Sciences, 20(9), 12–14. https://doi.org/10.3390/ijms20092181
Hughes, A. E., Bradley, D. T., Campbell, M., Lechner, J., Dash, D. P., Simpson, D. A., & Willoughby, C. E. (2011a). Mutation altering the miR-184 seed region causes familial keratoconus with cataract. American Journal of Human Genetics, 89(5), 628–633. https://doi.org/10.1016/j.ajhg.2011.09.014
Hughes, A. E., Bradley, D. T., Campbell, M., Lechner, J., Dash, D. P., Simpson, D. A., & Willoughby, C. E. (2011b). Mutation altering the miR-184 seed region causes familial keratoconus with cataract. American Journal of Human Genetics, 89(5), 628–633. https://doi.org/10.1016/j.ajhg.2011.09.014
Khan, Ahmed, Elareer, Junejo, Steinhoff, & Uddin. (2019). Role of miRNA-Regulated Cancer Stem Cells in the Pathogenesis of Human Malignancies. Cells, 8(8), 840. https://doi.org/10.3390/cells8080840
Kitamura, S., Maeda, T., & Yanagi, T. (2020). Vandetanib inhibits cell growth in EGFR-expressing cutaneous squamous cell carcinoma. Biochemical and Biophysical Research Communications, 531(3), 396–401. https://doi.org/10.1016/j.bbrc.2020.07.111
Lan, Y., Chen, H., Chen, J., & Lei, Q. (2014). Immunolocalization of Vimentin , Keratin 17 , Ki-67 , Involucrin , β -Catenin and E-Cadherin in Cutaneous Squamous Cell Carcinoma. 263–266. https://doi.org/10.1007/s12253-013-9690-5
Malzkorn, B., Wolter, M., Liesenberg, F., Grzendowski, M., Stühler, K., Meyer, H. E., & Reifenberger, G. (2010). Identification and functional characterization of microRNAs involved in the malignant progression of gliomas. Brain Pathology, 20(3), 539–550. https://doi.org/10.1111/j.1750-3639.2009.00328.x
Nagosa, S., Leesch, F., Putin, D., Bhattacharya, S., Altshuler, A., Serror, L., Amitai-Lange, A., Nasser, W., Aberdam, E., Rouleau, M., Tattikota, S. G., Poy, M. N., Aberdam, D., & Shalom-Feuerstein, R. (2017a). microRNA-184 Induces a Commitment Switch to Epidermal Differentiation. Stem Cell Reports, 9(6), 1991–2004. https://doi.org/10.1016/j.stemcr.2017.10.030
Nagosa, S., Leesch, F., Putin, D., Bhattacharya, S., Altshuler, A., Serror, L., Amitai-Lange, A., Nasser, W., Aberdam, E., Rouleau, M., Tattikota, S. G., Poy, M. N., Aberdam, D., & Shalom-Feuerstein, R. (2017b). microRNA-184 Induces a Commitment Switch to Epidermal Differentiation. Stem Cell Reports, 9(6), 1991–2004. https://doi.org/10.1016/j.stemcr.2017.10.030
Nasser, W., Amitai-Lange, A., Soteriou, D., Hanna, R., Tiosano, B., Fuchs, Y., & Shalom-Feuerstein, R. (2018). Corneal-Committed Cells Restore the Stem Cell Pool and Tissue Boundary following Injury. Cell Reports, 22(2), 323–331. https://doi.org/10.1016/j.celrep.2017.12.040
Nicolas, M., Wolfer, A., Raj, K., Kummer, J. A., Mill, P., van Noort, M., Hui, C. chung, Clevers, H., Dotto, G. P., & Radtke, F. (2003). Notch1 functions as a tumor suppressor in mouse skin. Nature Genetics, 33(3), 416–421. https://doi.org/10.1038/ng1099
Park, C. Y., Choi, Y. S., & McManus, M. T. (2010). Analysis of microRNA knockouts in mice. Human Molecular Genetics, 19(R2). https://doi.org/10.1093/hmg/ddq367
Sherwood, V., & Leigh, I. M. (2016). WNT Signaling in Cutaneous Squamous Cell Carcinoma : A Future Treatment Strategy ? Journal of Investigative Dermatology, December 2015, 1–8. https://doi.org/10.1016/j.jid.2016.05.108
Silpa, S. R., & V, C. (2013). A REVIEW ON SKIN CANCER. INTERNATIONAL RESEARCH JOURNAL OF PHARMACY, 4(8), 83–88. https://doi.org/10.7897/2230-8407.04814
Si, W., Shen, J., Zheng, H., & Fan, W. (2019). The role and mechanisms of action of microRNAs in cancer drug resistance. Clinical Epigenetics, 11(1), 1–24. https://doi.org/10.1186/s13148-018-0587-8
Smirnov, A., Anemona, L., Novelli, F., Piro, C. M., Annicchiarico-petruzzelli, M., Melino, G., & Candi, E. (n.d.). Unusual Skin Cancer b a.
Sobel, K., Tham, M., Stark, H. J., Stammer, H., Prätzel-Wunder, S., Bickenbach, J. R., & Boukamp, P. (2015). Wnt-3a-activated human fibroblasts promote human keratinocyte proliferation and matrix destruction. International Journal of Cancer, 136(12), 2786–2798. https://doi.org/10.1002/ijc.29336
Wong, T. S., Liu, X. B., Wong, B. Y. H., Ng, R. W. M., Yuen, A. P. W., & Wei, W. I. (2008). Mature miR-184 as potential oncogenic microRNA of squamous cell carcinoma of tongue. Clinical Cancer Research, 14(9), 2588–2592. https://doi.org/10.1158/1078-0432.CCR-07-0666

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XXXSupplementalMaterialTurovsky.pptx
Fig S1: Depletion of miR-184 enhances the formation of tumors with atypical features. (A) Genotyping was performed on tail DNA samples to identify wild type (WT) animals that carried 2 copies of Mir184 (+/+), knockouts (KO) (-/-) and heterozygous (+/-) mice. (B) Histology staining performed on skin paraffin sections of WT and KO tumors (see Fig. 1) showing keratin pearls that hallmark tumors isolated from WT animals, while large infundibular cyst structures that penetrated the deep dermis were prevalent in miR-184-KO tumors. (C) is quantification of (B). Data in represents 5 biological replicates. Fig. S2: Infection of miR-184 antagonist in skin squamous cell carcinoma cell line. (A) Schematic illustration of pLKO lenti-viral vector containing the antagonizing anti-miR-184 sequence (AM184) and a Histone 2B green fluorescent protein coding cassette (H2B-GFP) under the indicated promoters. (B) C12C20 SCC cells were infected with an empty (Ctl) vector or AM184 vector. GFP positive cells were sorted 3-4 days later by flow cytometry (B) and GFP expression was validated under the microscope (C). (D) Validation of miR-184 knockdown by AM was performed by TaqMan assay. Scale bars were 20 μm. Statistical significance was assessed by t-test (*p < 0.05) Fig S3: Differential expression of genes in the miR-184-null epidermis. (A) Heatmap of the differentially expressed genes across the wild type (WT) and knockout (KO) samples. The thresholds of |log2 (fold change)| and ≥ 1 padj ≤ 0.05 and maximal count ≥ 30 (reads) was applied. (B) Increased expression of hair keratinization associated genes was detected in KO samples.

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miR-184 represses β-catenin and behaves as a skin tumor suppressor

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Abstract

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Introduction

Materials and Methods

Cell culture

In vitro assays

Animal models

RNA extraction, real-time polymerase chain reactions and sequencing

Western Blot analysis, immunofluorescence and Immunohistochemistry

Statistical analysis

Results

miR-184-ablation leads to hyper stem cell activation and susceptibility to cancer

miR-184 inhibits neoplastic phenotype of human cutaneous squamous cell carcinoma

miR-184-repression of Wnt/β-catenin contributes to SCC phenotype

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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