Tumor suppressor function of miR-483-3p on squamous cell ...

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Cell Cycle 12:14, 2183–2193; July 15, 2013; © 2013 Landes Bioscience

Thomas Bertero,1,2 Isabelle Bourget-Ponzio,2,3 Alexandre Puissant,2,4 Agnès Loubat,2,5 Bernard Mari,1,2 Guerrino Meneguzzi,2,3 Patrick Auberger,2,4 Pascal Barbry,1,2,* Gilles Ponzio1,5 and Roger Rezzonico1,2,* 1 CNRS UMR 7275; IPMC; Valbonne, France; 2Université de Nice Sophia-Antipolis; Nice, France; 3Faculté de Médecine; IRCAN; CNRS UMR 7284; INSERM U 1081; Nice, France; 4INSERM U1065; C3M; Team 2 Bâtiment Universitaire Archimed; Nice, France; 5Faculté des Sciences; Institute of Biology Valrose; CNRS UMR7277; INSERM U1091; Nice, France

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he frequent alteration of miRNA expression in many cancers, together with our recent reports showing a robust accumulation of miR-483-3p at the final stage of skin wound healing, and targeting of CDC25A leading to an arrest of keratinocyte proliferation, led us to hypothesize that miR-483-3p could also be endowed with antitumoral properties. We tested that hypothesis by documenting the in vitro and in vivo impacts of miR-483-3p in squamous cell carcinoma (SCC) cells. miR-483-3p sensitized SCC cells to serum deprivation- and drug-induced apoptosis, thus exerting potent tumor suppressor activities. Its pro-apoptotic activity was mediated by a direct targeting of several anti-apoptotic genes, such as API5, BIRC5, and RAN. Interestingly, an in vivo delivery of miR-483-3p into subcutaneous SCC xenografts significantly hampered tumor growth. This effect was explained by an inhibition of cell proliferation and an increase of apoptosis. This argues for its further use as an adjuvant in the many instances of cancers characterized by a downregulation of miR-483-3p.

Keywords: microRNA, tumor suppressor, apoptosis, miR-483-3p, epidermis Submitted: 05/13/13 Accepted: 06/08/13 http://dx.doi.org/10.4161/cc.25330 *Correspondence to: Pascal Barbry and Roger Rezzonico; Email: [email protected] and [email protected]

www.landesbioscience.com

Introduction MicroRNAs (miRNAs) are small (~22 nt) noncoding RNAs that repress gene expression at the post-transcriptional level primarily by binding to 3' untranslated regions (3'UTR) of their target mRNAs, thereby inhibiting translation and/or leading to mRNA decay.1 The major determinant of miRNA target gene intermolecular

interaction is the “seed”, a stretch of 6–8 nucleotides localized at the 5' end of the mature miRNA. Therefore, a single miRNA can easily target several hundred genes and generate a complex gene regulatory network. The capacity to target different genes belonging to distinct signaling pathways can be useful to increase the efficiency of a specific miRNA. The roles of miRNAs have now been well established in various physiological and physiopathological processes, notably in cancer development. Numerous studies have not only characterized specific “miRNA signatures” associated with solid tumors, but they have even defined several of these “tumor-specific” miRNAs as either oncogenes (“oncomiRs”) or tumor suppressors.2-5 Functional studies evaluating the physiopathological implication of these miRNAs in tumorigenesis are still scarce, despite the fact that more information could pave the way to the development of new more powerful therapeutic approaches.6 Characterization of the molecular events controlling tissue repair is another promising way for the development of new anticancer therapeutic approaches. Indeed, by claiming that tumors could be considered either “over-healing wounds” or “wounds that do not heal”, Haddow’s and Dvorak’s seminal aphorisms have established a strong parallel between tumorigenesis and wound healing.7,8 This has been remarkably substantiated at many cellular and molecular levels.9 However, the “healing” process is not self-limiting

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©2013 Landes Bioscience. Do not distribute.

Tumor suppressor function of miR-483-3p on squamous cell carcinomas due to its pro-apoptotic properties

in cancer, leading to exacerbated cell proliferation, invasion, and metastasis.9 Thus, the identification of genes participating to the shutdown of the repair response could shed light on the mechanisms of tumorigenesis and even display antitumoral properties. Interestingly, we recently reported the strong and specific induction of miR483-3p in response to skin injury and demonstrated its potent anti-proliferative properties in keratinocytes at late stage of wound healing.10 We explained miR483-3p-mediated cell cycle arrest by the direct targeting of the CDC25A phosphatase that increases the tyrosine phosphorylation status of CDK4/6 cyclin-dependent kinases, thus preventing their association with cyclin D and blocking cells in early G1.11 These observations led us to hypothesize that miR-483-3p could exert antitumoral properties. This hypothesis was consistent with data showing that miR483-3p expression is decreased in gastric, nasopharyngeal, and hepatocellular carcinomas, even if its physiopathological implication in the development of these

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tumors was not proven.12-15 Conversely, high levels of miR-483-3p have been proposed to play an oncogenic role in tumors characterized by biallelic expression of its host gene IGF2 due to loss of imprinting (LOI) of the IGF2/H19 locus.16-20 In order to clarify these apparent discrepancies, we investigated the effects of miR-483-3p on SCC. We showed that miR-483-3p is indeed endowed of tumor suppressor properties. We demonstrated that, besides its anti-proliferative effect, miR-483-3p also sensitizes cells to apoptosis, and we identified new target genes mediating this pro-apoptotic action. We finally show that intra-tumoral delivery of miR-483-3p might be an adjuvant strategy for the treatment of cutaneous SCC, as well as for gastric, liver, and nasopharyngeal carcinomas in which miR-483-3p is downregulated. Results Ectopic expression of miR-483-3p inhibits tumor engraftment of SCC cells. To

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Figure 1. Effects of miR-483-3p on tumor growth. CAL27 cells were transfected with pre-miR-NC or pre-miR-483-3p (10 nM), trypsinized 48 h later and then either aggregated in spheroids that were embedded in 3D-gels (A) or injected (5 × 106 cells/mouse) subcutaneously on the flanks of nude mice (B). (A) Pictures are representative of CAL27 spheroids at 1, 5, and 10 d. Data are means ± SD of 10 spheroids per condition. (B) Tumor size was measured at 5, 8, 11, and 13 d post-injection. Data are means ± SD of 12 tumors per condition (**P < 0.001). Pictures are representative of CAL27 xenografts 14 d after inoculation.

determine the impact of miR-483-3p on tumor development, we first analyzed the effects of its ectopic expression on the three-dimensional growth capacity of CAL27 squamous cell carcinoma (SCC) cells using a multicellular tumor spheroids model used as a surrogate of tumor growth (Fig. 1A). CAL27 cells were transfected with a non-relevant pre-miR-NC or pre-miR-483-3p and allowed to aggregate under agitation to form spheroids that were embedded in a matrigel/collagen gel. miR-483-3p markedly reduced the growth of SCC spheroids compared with control (Fig. 1A), indicating that miR-483-3p delivered inhibitory signals for the development of tumor cells in a 3D environment. In addition, when miR-NC-transfected CAL27 cells were injected subcutaneously in nude mice to generate tumor xenografts, we observed tumors of 150– 180 mm3 8–13 d after grafting (Fig. 1B). In contrast, tumors generated from cells treated with miR-483-3p do not exceed a volume of 70–80 mm3. This experiment confirmed the marked inhibitory effect exerted by miR-483-3p on tumor engraftment in vivo. Mitochondrial contribution to the miR-483-3p-induced apoptosis in tumor cells. The antitumoral effect of miR483-3p may result from an inhibition of proliferation and/or an increase in cell death. We then analyzed its impact on cell survival. We observed that 16 h after serum starvation, miR-483-3ptransfected CAL27 cells massively died (Fig. 2A). This effect was inhibited by the addition of z-VAD-FMK, a pan-caspase inhibitor, indicative of an apoptotic process. Three independent approaches to measure apoptosis led essentially to the same conclusions: a collapse of mitochondrial membrane potential visualized by DiOC6 labeling, the activation of caspase 3, and the externalization of phosphatidylserine shown by Annexin V labeling were increased after miR-483-3p overexpression (Fig. 2A). As expected, the activation of caspase 3 was completely inhibited by z-VAD-FMK. These results demonstrate that the ectopic expression of miR-483-3p sensitizes CAL27 cells to apoptosis induced by serum deprivation. The increase in the number of apoptotic cells with reduced DIOC6 fluorescence

(Fig. 2A) suggests the implication of a mitochondrial pathway. To check this hypothesis, we analyzed the kinetics of activation of the effector caspases 3/7 and of the initiator caspases 8 and 9 relative to extrinsic and intrinsic apoptotic pathways, respectively (Fig. 2B). The results showed that in absence of serum, miR-483-3p induced the activation of caspase 3 and 9 in CAL27 cells, but did not alter the basal


activity of caspase 8, demonstrating the involvement of a mitochondrial cell death pathway. As expected, the activity of caspases 3 and 9 was completely inhibited in cells treated with z-VAD-FMK (Fig. 2B). To better characterize the molecular mechanisms responsible for the proapoptotic effect of miR-483-3p, we assessed its impact on the level of expression of anti-apoptotic BCL2 family

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members, including BCL2 itself, MCL1, and BCLX L (Fig. 2C). We observed that the overexpression of miR-483-3p significantly decreased the level of BCL2, and to a lesser extent that of BCLX L . The analysis of caspase 3/7 activity in two other OSCC cell lines, CAL33 and CAL60, confirmed that miR-483-3p sensitizes cancer cells to serum-starvationinduced apoptosis (Fig. 2D; Fig. S1).

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Figure 2. miR-483-3p induces mitochondrial-dependent apoptosis in SCC cells. (A–C) CAL27 cells were transfected with the pre-miR-483-3p or premiR-NC. After 48 h, cells were serum-starved for the indicated time in the absence or presence of z-VAD-FMK (50 μM). (A) Flow cytometry analysis of apoptotic cells after DiOC6, active caspase 3 and annexin V staining. Data are means ± SD of three independent experiments. (B) Time course of induction of the enzymatic activity of caspases 3/7, 9, and 8. The activity was measured after 0, 8, 16, 24, and 48 h of serum deprivation. Data are means ± SD of three independent experiments. (C) Western blot analysis of apoptosis regulator proteins expression. HSP60 was used as a loading control. (D) CAL33 and CAL60 OSCC cells were transfected, serum-starved, and assessed for the enzymatic activity of caspases 3/7 as in (B). Data are means ± SD of three independent experiments.

miR-483-3p potentiates drug-induced apoptosis. Then we wondered whether miR-483-3p also altered cell death sensitivity against chemotherapeutic reagents. To do so we measured the impact of miR483-3p on caspase 3/7 activity in SCC cells treated with sub-optimal doses of drugs commonly used in chemotherapy, such as etoposide, cisplatin, and camptothecin, in the presence of serum. As shown in Figure 3A, miR-483-3p overexpression resulted in a specific increase in caspase 3/7 activity in CAL27, CAL33, and CAL60 cells treated with etoposide, cisplatin, and camptothecin compared with control condition (miR-NC). These potentiations ranged between 1.4- and 2-fold of the effects of drugs alone. Since CAL60 cells express a higher level of miR-483-3p (Fig. S2), we then evaluated the effects of its blocking using an anti-miR-483-3p (LNA-483-3p) on drug-induced effector caspase activation (Fig. 3B). We observed that the inhibition of endogenous miR-483-3p significantly decreased the pro-apoptotic effects of etoposide and cisplatin and, to a lesser extent, that of camptothecin. Identification and validation of miR483-3p target genes. We then searched for putative target genes that could mediate the pro-apoptotic action of miR-483-3p. Analysis of the 3'UTR of the miR-483-3pdownregulated genes after expression of miR-483-3p in keratinocytes10 allowed us to identify three putative target genes that could play a role in miR-483-3pinduced apoptosis: the apoptosis inhibitor proteins, API5 and BIRC5 (survivin), and the small GTPase family member RAN involved in nuclear transport (Fig. 4A).

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These target genes were validated after a cloning of their 3'-UTR, containing the putative miR-483-3p binding sites (Fig. 4A), into the psiCHECK-2 vector downstream to a luciferase reporter gene. miR483-3p inhibited the luciferase activity of BIRC5, RAN, and API5 constructs from 40–75% (Fig. 4B). The mutation of miR483-3p binding sites in these constructs completely abolished the inhibitory action of miR-483-3p. In addition, western blot analyses showed that the expressions of API5, RAN, and BIRC5 were significantly reduced in CAL27, CAL33, and CAL60 cells treated with miR-483-3p (Fig. 4C). Conversely, antagonizing endogenous miR-483-3p, in CAL60 cells, increased the expression of API5, RAN, and BIRC5 (Fig. 4D). Taken together, these data indicate that API5, BIRC5, and RAN represent bona fide target genes of miR-483-3p that could play a main role in miR-483-3p-induced apoptosis. Role of miR-483-3p targets in SCC cells apoptosis. The impact of API5, BIRC5, and RAN invalidation on SCC cells apoptosis was assessed using specific siRNAs. We showed that BIRC5 and RAN knockdown decreased the expression of BCL2 and stimulated caspase 3 activation following serum starvation as shown by the cleavage of PARP-1 (Fig. 5A) and caspase 3/7 enzymatic assay (Fig. 5B), thus mimicking the action of miR-483-3p. These effects were abolished in the presence of z-VAD-FMK. Invalidation of API5 had no effect on caspase 3 activity. We recently reported that targeting of CDC25A by miR483-3p inhibited cell cycle progression of

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wounded keratinocytes.10 Since CDC25A has also been shown to control apoptosis,21,22 we assessed its role in the mediation of miR-483-3p-induced cell death. To test this hypothesis, we used a CAL27 cell line that stably express a CDC25A form lacking its 3'UTR, and thus insensitive to miR-483-3p. miR-483-3p still decreased the expression of API5, BIRC5, and RAN and activates effector caspases in these cells (Fig. S3). The pro-apoptotic effect observed here is thus independent of miR-483-3p’s effect on CDC25A. Taken together, these results suggest that the targeting of BIRC5 and RAN is crucial in the mediation of miR-483-3p pro-apoptotic properties. Effects of the intra-tumoral delivery of miR-483-3p. The antitumoral therapeutic potential of miR-483-3p was tested after in vivo delivery on SCC xenografts of CAL33 cells that were subcutaneously implanted in nude mice. When tumors reached a volume of about 100 mm3, three successive intra-tumoral injections at three-day intervals of miR-483-3p or miR-NC complexed to PEI (polyethylenimine) nanoparticles were delivered to the tumors.23 Monitoring of the tumor size during 10 d demonstrated that miR483-3p significantly hampered tumor development (Fig. 6A). We observed a marked reduction of tumor growth after the second miR-483-3p injection. After the third injection, it reduced tumor size by 50%. Ectopic expression of pre-miR-483-3p increased the expression of miR-483-3p by 50 fold (Fig. 6B). To ensure that miR-483-3p was truly delivered intracellularly in tumors, we analyzed the

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Figure 3. miR-483-3p favors drug-induced apoptosis. CAL27, CAL33 and CAL60 cells were transfected with pre-miR-483-3p or pre-miR-NC (10 nM) (A) or with anti-miR-483-3p (LNA-483-3p) or anti-miR-control (LNA-159s) (20 nM) (B). After 48 h, cells were incubated for 24 h in the presence of camptothecin (CPT, 1 μM), cisplatin (CIS, 20 μM), or etoposide (ETO, 20 μM) before assaying for caspase 3/7 activity. Data are means ± SD of three independent experiments (*P < 0.01).

consequences of its in vivo delivery on the expression of target genes transcripts and on apoptosis in tumor homogenates. Intra-tumoral injections of miR-483-3p decreased by 30–40% the expression of BIRC5, RAN, and CDC25A mRNA levels (Fig. 6C), demonstrating that these genes were efficiently targeted by miR-483-3p in vivo. Increased caspase 3 activity confirmed that miR-483-3p induced apoptotic cell death in tumors (Fig. 6D). In addition, the expression of cell proliferation and apoptosis markers were quantified in miRNA-treated tumors using high-speed immunohistofluorescence microscopy (Fig. 6E and F). miR-483-3p inhibited cell proliferation by 25–30%, as assessed by PCNA (proliferating cell nuclear antigen) labeling, while it stimulated the amount of apoptotic cells detected by TUNEL staining by 30%. These effects probably correspond to a low estimate of the effect, considering the non-homogeneous delivery of miR483-3p in tumors. The antitumoral properties of miR483-3p thus involve, simultaneously and


independently, an inhibition of cell proliferation and an induction of apoptosis. Both effects make this miRNA a suitable molecule for adjuvant therapy of cancers characterized by a decreased expression of miR-483-3p. Discussion We recently depicted the anti-proliferative action of miR-483-3p in healing keratinocytes due to CDC25A knockdown.10,11 We further demonstrate here that the miR483-3p-mediated inhibition of CDC25A expression also occurs in vitro and in vivo in tumor cells. We have previously demonstrated that miR-483-3p-mediated inhibition of CDC25A increases the tyrosine phosphorylation status of CDK4/6 cyclindependent kinases, which, in turn, abolished CDK4/6 capacity to associate with D-type cyclins. This prevented CDK4/6 kinase activation and finally sequestered cells in early G1. This regulatory process represents a paradigm for coupling miRNA-mediated CDC25A invalidation to cell cycle control, which can operate

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with several anti-proliferative miRNAs targeting CDC25A, including miR-15a, miR-16, miR-21, miR-125b, miR-424, miR-449a/b/c, miR-503, let-7, and the miR-497/miR-195 cluster.24-32 From that perspective, repression of CDC25A by miRNAs is a significant component of cell cycle control and fits well with the role of specific miRNAs in differentiation programs. Thus, miR-424/miR-503 and miR449, which play roles in myogenesis and multiciliogenesis, respectively, have been shown to promote cell quiescence, notably through CDC25A repression.28,29,33,34 Moreover, most of the CDC25A-targeting miRNAs are repressed in hyperproliferative diseases. This is the case of miR-15a in polycystic liver disease,25,35 and of miR15a/miR-16,36 miR-125b,30 miR-424,37 miR-497/miR-195,32 or miR-449a/b38,39 in cancers, in which they exert tumor suppressor functions through silencing of multiple oncogenic targets. We also demonstrate here that miR483-3p is not only an anti-proliferative miRNA, since it sensitizes tumor cells to several pro-apoptotic stimuli, such as

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Figure 4. Validation of miR-483-3p pro-apoptotic target genes. (A) Sequence of putative miR-483-3p binding sites in the 3'-UTR of human BIRC5, RAN and API5. (B) Luciferase reporter assays of wild-type (WT) and mutated (MUT) 3'-UTR sequences of BIRC5, API5, and RAN in the presence of pre-miR-NC or pre-miR-483-3p. Data are means ± SD of normalized luciferase activity from three independent experiments. Statistically significant differences are indicated (**P < 0.001). (C) Western blot analysis of API5, RAN, and BIRC5 expression in CAL27, CAL33, and CAL60 cells transfected with pre-miR-483-3p or control pre-miR-NC for 48 h. (D) CAL60 cells transfected with LNA-483-3p or LNA-159s were harvested after 72 h, and expression of API5, RAN, and BIRC5 was analyzed by western blot. HSP60 was used as a loading control.

serum starvation or chemotherapeutic drugs. These observations are in total agreement with the notion that functions of miRNAs often become pronounced under conditions of stress, making them potential targets for replacement therapies in various diseases, including cancer.40 Our first working hypothesis was that the direct targeting of CDC25A by miR483-3p could explain the pro-apoptotic properties of this miRNA. This was in line with the fact that: (1) the knockdown of CDC25A sensitizes keratinocytes to UV-induced apoptosis;21 (2) the pharmacological inhibition of CDC25A phosphatase activity inhibits the growth of tumor spheroids by promoting apoptosis.41 But CDC25A is also frequently overexpressed in different types of cancers, where it participates in tumor cell survival and resistance to chemotherapy.22,42,43 The expression of CDC25A promotes cell survival and resistance to cisplatin-induced apoptosis via induction of NFκB.44 However, in our hands, SCC cells expressing a miR-483-3p-resistant form of CDC25A died from apoptosis following serum removal (Fig. S3), suggesting that other target genes were indeed involved in the mediation of the miR-483-3p proapoptotic action. We finally identified survivin (BIRC5), RAN, and API5 as new targets of miR483-3p. Survivin is a well-known inhibitor of apoptosis that is overexpressed

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in SCC. It protects keratinocytes from UV-induced apoptosis,45 and it is targeted by multiple miRNAs with tumor-suppressive functions. Overexpression of RAN is associated with poor patient outcome in ovarian, breast, and lung cancers, and its knockdown results in mitotic defects and apoptosis.46-48 Moreover, RAN and survivin form complexes that were shown to be crucial for mitotic spindle formation and chromosome segregation in tumor cells.49 Interestingly, RAN is also targeted by miR-203, whose expression is decreased in healing epidermis, thus favoring keratinocytes migration and proliferation.50 Finally, despite that API5 was previously shown (1) to protect cells from growth factor deprivation- or drug-induced apoptosis51,52 and (2) to be targeted by other miRNAs,53,54 it seemed non-essential for the pro-apoptotic effects of miR-483-3p. Indeed, in our hands, API5 knockdown did not affect BCL2 expression and did not induce caspase 3 activity following serum removal. Further experiments would be needed to clearly establish the each target gene contribution in the mediation of the antitumoral action of miR-483-3p. Our study represents a proof of concept that identification of a mechanism involved in the arrest of wound healing can represent a relevant target for the development of novel anticancer strategies. The ectopic expression of miR-483-3p, a mediator of wound-healing arrest in

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keratinocytes, markedly sensitized SCC cells to pro-apoptotic stimuli in vitro and showed a potent antitumoral effect when delivered in vivo. Other “wound-healing miRNAs” such as miR-198 and miR-203 could also fulfill these requisites. miR-198 and miR-203 are downregulated following skin injury and allow the keratinocyte migration and proliferation necessary to wound closure,50,55 suggesting that an ectopic expression of these miRNAs could limit tumorigenesis. Indeed, miR-203 has recently been shown to be a tumor suppressor in basal cell carcinoma,56 and decreased miR-198 expression is observed in hepatocellular carcinomas, where it favors tumor cells growth and invasion.57-59 Other opportunities for the development of replacement therapies also come from miRNAs that are dysregulated in pretumoral lesions such as chronic wounds or ulcers. This could be particularly challenging for patients suffering from recessive dystrophic epidermolysis bullosa, a genodermatosis characterized by severe erosive skin lesions with an elevated risk of invasive SCC development.60 Altered methylation of the IGF2/H19 locus has been linked to programmed changes in IGF2 expression, the host gene of miR-483-3p. Consequently, miR-483-3p is overexpressed in different cancers, such as childhood Wilms tumors, adrenocortical tumors, mesotheliomas, and colorectal, pancreatic, and

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Figure 5. Functional analysis of miR-483-3p pro-apoptotic target genes. (A and B) CAL27 cells were transfected with API5, BIRC5, RAN or control siRNAs. Forty-eight h later, cells were serum-deprived for 24 h in the presence or absence of z-VAD-FMK (50 μM). Cells were analyzed by western blot for the expression of cleaved PARP-1, BCL2, API5, BIRC5, RAN, and HSP60 (A), and assayed for the activity of caspase 3/7 (B). Similar results were obtained with two different siRNA sequences. Data are means ± SD of three independent experiments.

©2013 Landes Bioscience. Do not distribute. Figure 6. Effects of in vivo delivery of miR-483-3p on tumor growth. (A) Subcutaneous xenografts were established in nude mice by injection of CAL33 cells as described in “Materials and Methods”. After 7 d, the tumors were injected three times at 3 d intervals with pre-miR-483-3p or pre-miRNC coupled to PEI nanoparticles, and tumor volume was assessed over time. Data are means ± SD of 10 tumors/group (**P < 0.005). (B) qPCR analysis of miR-483-3p expression normalized to RNU44 level in tumors injected with miR-NC or miR-483-3p. (C) Relative expression of BIRC5, API5, RAN, and CDC25A mRNA levels in CAL33 xenografts treated with miR-483-3p and miR-NC. (D) Effects of intra-tumoral injection of miR-483-3p on caspase 3/7 activity. (B–D) Data are means ± SD of eight biopsies/group (**P < 0.01, *P < 0.05). (E) Pictures are representive of immunohistofluorescence analysis of PCNA and TUNEL levels in whole-xenograft sections. (F) Normalized quantification of PCNA and TUNEL immunofluorescence labelings of whole tumor sections (n = 24, *P < 0.01).


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Furthermore, while miR-483-3p expression is not altered in cutaneous SCC, one can speculate that its topical delivery might also be of particular interest to slow down the development of such tumors. Materials and Methods Cell culture and transfection. The human CAL27, CAL33, and CAL60 oral squamous cell carcinoma cell lines70,71 were kindly provided by Dr JL Fischel (Centre Antoine Lacassagne), and the COS-7 monkey kidney cell line was from ATCC. Cells were grown in DMEM containing 10% fetal calf serum (Perbio Science AB). Pre-miRNAs (pre-miR-483-3p and negative control pre-miR-NC1), antimiRNAs oligonucleotides (LNA-159s and LNA-483-3p), and ON-Targetplus siRNAs (for BIRC5, API5, RAN, and irrelevant control [Ctl]) were purchased from Ambion, Exiqon (Vedbaek) and Dharmacon (Thermo Fisher Scientific), respectively. Cells were plated and transfected 24 h later at 30–50% confluency, with pre-miRNAs (10 nM), anti-miRNAs (20 nM), or siRNAs (25 nM) using Lipofectamin™ RNAiMAX reagent (Life Technologies). CAL27 cells stably expressing WT-CDC25A were generated by transfection of pcDNA3.1 construct (gift from Dr S Manenti) using Fugene reagent (Promega) and selected in medium containing 1 mg/ml of G418 (Life Technologies). FACS analysis of apoptosis. Cells were stimulated, harvested, and washed with phosphate-buffered saline (PBS), and stained with an activated caspase 3 antibody (caspase 3 kit, Roche) or with FITC-labeled annexin V reagent (annexin V FLUOS staining kit, Roche) and propidium iodide (PI) for 15 min according to the manufacturer protocols. Apoptotic cells correspond to annexin V-positive/PI-positive and annexin V-positive/PI-negative populations. To determine the mitochondrial membrane depolarization, cells were incubated with 50 nM of 3,3-dihexyloxacarbocyanine (DiOC6) (Molecular Probes) and propidium iodide for 15 min at 37 °C. After washing, data were acquired on a FACScalibur flow cytometer and

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analyzed using the CellQuest Pro software (Becton-Dickinson). Caspase assays. The activation of initiator caspases 8 and 9 and executioner caspases 3 and 7 in SCC cells was determined using the Caspase Glo 3/7, Caspase Glo 8, and Caspase Glo 9 assay kits (Promega) according to manufacturer’s instructions. Cell and tumor lysates were incubated in triplicate in 96-well plates and luminescence was quantified after 1 h of incubation with the caspase substrates. In some experiments, cells were treated with the z-VAD-FMK (carbobenzoxyvalyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone) cell-permeable pan caspase inhibitor (Bachem) to inhibit induction of apoptosis. Antibodies. The rabbit polyclonal antibody (pAb) to API5 was from Abcam. Rabbit pAbs to BIRC5, BCL2, BCL-xL, MCL1, and PARP were from Cell Signaling Technology. The mouse monoclonal antibody (mAb) to CDC25A and goat pAbs to HSP60 and RAN were purchased from Santa Cruz Biotechnology (Santa Cruz). The secondary peroxidaseconjugated pAb were from Dakopats. miRNA and gene expression analyses by real-time qPCR. Total RNA was isolated with TRIzol™ reagent (Life Technologies) according to the manufacturer’s instructions. Expression of mature hsa-miR-483-3p was evaluated using TaqMan MicroRNA Assays (Applied Biosystems) and normalized to RNU44. For gene expression, RNAs were retrotranscribed with the Superscript II RT kit (Invitrogen) and PCR performed using SYBR Green Master mix II (Applied). Gene expression levels were normalized to RPLP0 level. All reactions were done in triplicate using ABI PRISM 7900HT Sequence detection system (Applied) and expression levels calculated using the comparative CT method (2−ΔΔCT ). Plasmid constructs, site-directed mutagenesis, and luciferase assays. cDNA fragments corresponding to human BIRC5, API5, and RAN 3'-UTR mRNAs were cloned into the XhoI and NotI restriction sites downstream from the Renilla luciferase gene of psiCHECK-2 vector (Promega) using the following primers: BIRC5-F 5'-CCGCTCGAGG AGCCTGTGCT GTGGGCAGG-3'

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hepatocellular carcinomas.16-20 Functional studies have reported a protumoral role of miR-483-3p that was explained by the direct targeting of the apoptotic effector BBC3/PUMA.16,17 This is clearly not the case in normal and tumor keratinocytes, where miR-483-3p is always anti-proliferative and pro-apoptotic (here and in refs. 10 and 11). This tumor suppressor function is consistent with many reports showing its decreased expression in gastric, nasopharyngeal, and hepatitis B virus-associated hepatocellular carcinomas.12-15 One explanation for these discrepancies is that miR-483-3p could exert opposite effects on the development of cancer, according to its cellular context. A similar situation stands for the miR-29 family, which exhibits tumor suppressive functions in lung cancer, chronic lymphocytic leukemia, and cholangio-carcinoma, because it targets oncogenes including Tcl1, Mcl-1, and the DNA methyltransferases 3A and 3B, while it is oncogenic in breast tumors due to the inhibition of ZFP36.61-65 miR31 is another example of miRNA that exerts distinct functions in tumor development depending on tumor stage, tissue type, and/or genetic context. Indeed, miR-31 inhibits the formation of breast cancer metastases via the concomitant repression of ITGA5 (integrin α5), RDX (radixin), and RhoA,66,67 but it promotes primary tumor growth in head-and-neck and lung SCC.68,69 Consequently, additional investigations are needed to better characterize miR-483-3p biological functions and, notably, the identification of its downstream effectors in various cancers. In conclusion, our findings demonstrated that miR-483-3p exerts tumor suppressor functions on SCC through repression of multiple targets, including the cell cycle regulator CDC25A and the anti-apoptotic proteins BIRC5 and RAN. Currently, miRNA replacement therapies looks particularly appropriate for the treatment of different pathologies, mainly because, due to their small size, miRNA are poorly antigenic and easy to deliver into cells or tissues without using potentially hazardous viral approaches.6 In this context, the restoration of miR-483-3p in cancers in which its expression is decreased could provide a promising future adjuvant antitumoral strategy.


free access to food. Six-week-old female immune-deficient NMRI mice (Janvier) were anesthetized by isoflurane inhalation and inoculated subcutaneously with CAL27 or CAL33 human SCC cells (5 × 106 cells per mouse in 50% Matrigel). For tumor engraftment experiments, SCC cells were transfected with premiR-NC or premiR-483-3p (10 nM) and injected 48 h after transfection. The growth tumor curves were determined by measuring the tumor size with calipers and calculating their volume using the equation V = (L × W2)/2. For intra-tumoral delivery of miR483-3p, subcutaneous xenografts of CAL33 cells were established. One week later, when tumors reached a size of ~100 mm3, they were injected three times at 3-d intervals (d7, d10, and d13) with 25 μl of a solution containing 0.75 nmol of premiRNAs complexed with nanoparticles of in vivo jet PEI (Polyplus Inc) in 5% glucose. After 16 d, mice were killed by CO2 inhalation. Tumors were harvested and aliquots were either snap-frozen for further RNA and protein extractions or fixed in formalin and embedded in paraffin (FFPE) for immunohistological analyses. Immunofluorescence histology. FFPE tumor sections (5 μm) were dewaxed and labeled with an anti-PCNA antibody to detect proliferating cells (AbCys, Paris) or for TUNEL to detect apoptotic cells using the in situ cell death detection kit (Roche) according to the manufacturers’ instructions. Labelings were revealed with an Alexa fluor 488-conjugated anti-mouse antibody (Invitrogen), and nuclei were counter-stained with DAPI. Coverslips were mounted on glass slides and images acquired with the AxioVision software on a Axio Observer Z1 motorized high-speed epifluorescence microscope equiped with a EMCCD video camera (Cascade II: 1024) (Carl Zeiss S.A.S.). The relative fluorescence of the Alexa fluor 488 signals of PCNA and TUNEL labelings was quantified in three different sections spaced from 500 μm of each tumor and normalized to the DAPI fluorescence. Statistical analysis. Statistical evaluations were performed by Student t test for paired data, and data were considered significant at a P value less than 0.05.

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Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed. Acknowledgments

This work was supported by funds from the Association pour la Recherche sur le Cancer (ARC, Grant 5420). Thomas Bertero was a recipient of a doctoral contract from the Ministry of Higher Education and Research. Supplemental Materials

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BIRC5-R 5'-ATAAGAATGC GGCCGCGGCT TGCTGGTCTC TTCTGGGC-3' API5-F 5'-CCGCTCGAGG GGATGCTCTC TCGGTTTGCT TTTG-3' API5-R 5'-ATAAGAATGC GGCCGCGCAG TGTAGAGGTA ATGGTAATGA AGAC-3' RAN-F 5'-CCGCTCGAGC AGACAACTGC TCTCCCGGAT GAG-3', and RAN-R 5'-ATAAGAATGC GGCCGCCGAA AAGGAATGGG AATGACAGTA ACAAAC-3'. Luciferase assays were performed in COS-7 cells as previously described.10 Mutagenesis of the putative miR-483-3p binding sites was performed using the QuickChange multisite-directed mutagenesis kit from Stratagene (Agilent) according to the manufacturer’s protocol. Mutagenesis of 3' UTRs was performed using the following primers, in which “seed”-interacting sequences are in boldface and mutated bases are underscored: BIRC5-MUT 5'-CAGGTTCTCT AAGTTGGTG AGCAGTCTGG GAAGGG-3' API5-MUT 5'-CAACAGATGG GGGGTTTGTG CACTCTTTAA TGTCATGGGC-3', and RAN-MUT 5'-GCTGAAGGAG ATGAGTGGGC TTCGGTGAGA ATGTGGCAG-3'. Three-dimensional spheroid growth of SCC cells. CAL27 cells were transfected with pre-miR-483-3p or premiRNC, trypsinized 48 h later, and allowed to aggregate under agitating non-adherent conditions to form spheroids.72 The aggregates of cells were embedded in a Matrigel/type I collagen gel in MatTek 35 mm dishes (MatTek) and maintained in complete media over a period of time of 11 d. Pictures were taken at different times and the size of spheroids was measured using the ImageJ software. Subcutaneous SCC xenografts and intra-tumoral injections. Animal experiments were performed in accordance with the Declaration of Helsinki and were approved by a local ethical committee. Animals were maintained in a temperature-controlled facility (22 °C) on a 12-h light/dark cycle and were given

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