Targeting CDC7 sensitizes resistance melanoma cells to BRAFV600E-specific inhibitor by blocking the CDC7/MCM2-7 pathway

Although the utilization of selective BRAFV600E inhibitors is associated with improved overall survival in patients with metastatic melanoma, a growing challenge of drug resistance has emerged. CDC7 has been shown to be overexpressed and associated with poor prognosis in various cancers including melanoma. Thus, we aimed to elucidate the biological role of CDC7 in promoting Vemurafenib resistance and the anticipated benefits of dual targeting of BRAFV600E and CDC7 in melanoma cells. We performed exosomes-associated microRNA profiling and functional assays to determine the role of CDC7 in drug resistance using Vemurafenib-sensitive and resistant melanoma cells. Our results demonstrated that Vemurafenib-resistant cells exhibited a persistent expression of CDC7 in addition to prolonged activity of MCM2 compared to drug-sensitive cells. Reconstitution of miR-3613-3p in resistant cells downregulated CDC7 expression and reduced the number of colonies. Treatment of cells with low concentrations of CDC7 inhibitor TAK-931 sensitized resistant cells to Vemurafenib and reduced the number of cell colonies. Taken together, CDC7 overexpression and downregulation of miR-3613-3p were associated with Vemurafenib resistance in BRAFV600E- bearing melanoma cells. Dual targeting of CDC7 and BRAFV600E reduced the development of resistance against Vemurafenib. Further studies are warranted to investigate the clinical effect of targeting CDC7 in metastatic melanoma.

50% of melanoma patients acquire resistance against Vemurafenib, limiting its anti-tumor efficacy and reviving tumor progression within 6 to 8 months of treatment 12 . Accordingly, there is a critical need to understand the molecular mechanisms underlying resistance against BRAF V600E -targeted therapy, in an attempt to develop novel targeted therapies that circumvent the challenge of drug resistance in metastatic melanoma and maximize patients' survival.
In addition to other suggested mechanisms of resistance, acquired resistance to BRAF V600E inhibitors is predominantly linked to MAPK pathway reactivation 9,13 . Interestingly, overexpression of specific microRNAs (miRs) restore the sensitivity of resistant melanoma cells by downregulating their target genes, which are closely related to acquired resistance to BRAF V600E inhibition 14 . This makes miRs as attractive therapeutic targets in resistant melanomas. Due to the genetic complexity of melanomas and the concomitant activation of multiple signaling pathways, combinational therapy has been suggested to enhance the efficacy of treatment 15 .
Cell cycle dysregulation has been shown to mediate resistance of melanoma cells to anti-cancer drugs comprising MAPK inhibitors 10 . One of the most crucial regulator of the cell cycle is the cell division cycle 7 (CDC7) 16 . CDC7 is a highly conserved serine-threonine protein kinase, which is required for initiating the replication machinery. It particularly regulates G1/S phase transition, which is fundamental in cells proliferation, and its deregulation leads to oncogenesis 16 . CDC7 phosphorylates MCM2-7 for the helicase activation by uncoiling the double-stranded DNA as an initial step in DNA replication 17,18 . CDC7 that has been shown to be overexpressed in various cancers 16 was interrelated with poor prognosis including melanoma 18 . Although various CDC7 pharmacological inhibitors have been developed exhibiting substantial antitumor activity as a single agent, no studies have been conducted on CDC7 inhibition as an alternative targeted therapy in BRAF inhibitors-resistant melanoma cells. Recently, the safety, tolerability, and activity of CDC7-specific inhibitor TAK-931 was evaluated in patients with solid tumors and showed a clinical promise 19 .
In this study, resistant A375-NRAS Q61K (mutant NRAS) and WM983B-BR (wild-type NRAS) melanoma cell lines were used, which harbor BRAF V600E , and exhibit resistance to BRAF inhibitor, Vemurafenib, compared to its parental cells. We utilized recent CDC7 inhibitor, TAK-931, as a therapeutic option to circumvent the challenge of developed Vemurafenib resistance in melanoma cells.

Differential sensitivity of melanoma cells towards BRAF V600E -specific inhibitor Vemurafenib.
To distinguish the resistant phenotype of melanoma cells against selective inhibition of BRAF V600E by Vemurafenib, parental A375 (A375-P) & WM983B (WM983B-P), and resistant A375-NRAS Q61K (A375-R) & WM983B-BR (WM983B-R) were used in this study. As initial step, cell viability assay was performed to assess the differential sensitivity of melanoma cells to Vemurafenib treatment. Figure 1A showed that the concentrations of 0.1 and 2.5 µM Vemurafenib were enough to cause 50% inhibition of cell proliferation in A375-P and WM983B-P, respectively. In contrast, A375-R and WM983B-R cells were insensitive to the increase of Vemurafenib concentrations up to 2.5 and 5 µM, respectively. To further characterize the differential response of the parental versus resistant melanoma cells, the effect of Vemurafenib on MAPK signaling pathway was evaluated by Western blot analysis at different time intervals. As shown in Fig. 1B and Supplementary Fig. 1, the basal level of phospho-ERK1/2 (p-ERK1/2) was detected in both parental and resistant non-treated melanoma cells. However, ERK1/2 activity in A375-P and WM983B-P cells showed a complete inhibition at all selected time points compared to a persistent ERK1/2 activation in A375-R and WM983B-R cells over the course of 24 h treatment. In addition, phosphorylated Akt (p-Akt) showed a reduction in its activity after 24 h of treatment in A375-P and WM983B-P parental versus resistant A375-R and WM983B-R cells. miR-3613-3p is downregulated in A375-R cells-associated exosomes and targets CDC7 expression. We did microarray analysis for exosomes isolated from the conditioned media procured from A375-R regarding its parental cells and we identified a number of up-and down-regulated miRs (Table 1). miR-3613-3p was identified as one of top listed downregulated miRs (~8.5 folds). It has been reported that exosomes contribute to tumor progression, metastasis and drug resistance via transferring fully functional cargo including miRs into recipient cells in tumor microenvironment 20 . Therefore, we isolated miRs from exosomes and from cells to examine whether exosomes can differentially upload miRs in their cargo to promote drug resistance. Next, we validated the expression of miR-3613-3p in the 2 pairs of parental and resistant (A375 & WM983B) melanoma exosomes in addition to their respected cells using Real-Time PCR analysis (Fig. 1C). There was no statistical difference between A375-P and -R on cellular level (p = 0.126); however, a significant downregulation (−6.8-fold, p = 0.0003) was recorded for miR-3613-3p in A375-R exosomes. However, miR-3613-3p was upregulated (p < 0.001) on exosomal and cellular levels of WM983B-R by ~9.5 folds and ~19.5 folds, respectively. Using bioinformatic analyses, we identified several targets for miR-3613-3p; and CDC7 was one of these candidates (Fig. 1D). Obviously, this step was followed by examining the expression of CDC7 in A375 and WM983B cells. Surprisingly, the endogenous expression of CDC7 was higher in resistant versus parental cells in case of A375 cells while the signal of CDC7 was lower in parental and completely lost in resistant WM983B-R cells (Fig. 1E & Supplementary Fig. 2,A). Treatment of cells over 48 h with 2.5 µM Vemurafenib inhibited the expression of CDC7 in parental cells, compared to persistent expression in resistant cells ( Fig. 2A & Supplementary Fig. 2B). Since WM983B-R cells have very low expression of CDC7, we utilized A735 cells in the rest of our experiments. To examine the role of miR-3613-3p in the context of drug resistance in melanoma, resistant cells were transfected with either miR-3613-3p mimic at 25 and 50 nM concentrations or scrambled siRNA as a negative control and the success of transfection was validated by qPCR analysis (Fig. 2B). Approximately 48 h post-transfection, protein lysates were collected and Western blot analysis was conducted. As shown in Fig. 2C and Supplementary Fig. 3, reconstitution of miR-3613-3p in A375-R cells showed a slight decrease in CDC7 expression at 25 nM of the miR mimic and reached the maximum inhibition (62%) at 50 nM concentration. While miR-3613-3p overexpressed in A375-R using 50 nM mimic concentration, the activity of ERK1/2 was reduced compared to the control cells. Cell proliferation of transfected cells was assessed by clonogenic assay to determine the effect of miR-3613-3p in A375-R cells. Twenty-four hour after a single transfection of cells with 50 nM mimic, cells were plated at low density and monitored over 10 days. The results of long-term growth assay indicated that overexpression of miR-3613-3p caused colony growth arrest in A375-R cells by 21% (P < 0.01) as compared to scrambled cells (Fig. 2D).

Pharmacological inhibition of CDC7 by its specific inhibitor TAK-931.
In an attempt to further understand the contribution of CDC7 to Vemurafenib resistance, CDC7 expression was pharmacologically inhibited by selective CDC7 inhibitor, TAK-931. Cell viability assay was performed to assess the differential sensitivity of melanoma cells to TAK-931. Accordingly, cells were exposed to increasing concentrations of TAK-931 ranging from 10 to 1000 nM for 5 consecutive days. The results revealed that A375-P cells and A375-R showed a concentration-dependent proliferation inhibition when compared to non-treated cells. However, the IC 50 for TAK-931 was recorded at 150 nM in A375-R and 350 nM in A375-P (Fig. 3A). WM983B cells showed a lower response to TAK-931 compared to A375 cells because WM983-R cells have very low expression of CDC7 (Fig. 3B). Accordingly, we aimed to investigate whether the suppression of CDC7 could be an alternative therapy for resistant melanoma cells to overcome Vemurafenib resistance. Time course of drug treatment up to 48 h was performed for A375-R cells with either Vemurafenib (2.5 µM) or TAK-931 (500 and 1000 nM), and the difference of CDC7 expression and MCM2/ERK1/2 activities were assessed. As expected, CDC7 expression, and p-MCM-2 and p-ERK were persisitant after treating cells with Vemurafenib ( Fig. 3C & Supplementary Fig. 4). Remarkably, treating A375-R cells with TAK-931 resulted in inhibition of CDC7 expression as early as 1 h after treaement and this effect was lasting to 24 h. In parallel, p-MCM-2 showed dramatic decrease as early as 1 h up to 48 h after TAK-931 treatment. Moreover, TAK-931 inhibited ERK1/2 activity starting from 6 h up 24 h of treatment at 500 nM concentration. Notably, ceaved PARP1 was mostly recogized after 48 h of treatment (Fig. 3C).  4A). In addition, clonogenic assay was performed to assess the effect of CDC7 inhibition over a longer time period. Resistant cells were plated at low density then exposed to increasing concentrations of either Vemurafenib (0.1 to 10 µM), or TAK-931 (10 to 1000 nM), and monitored for 10 days. The treatment of A375-R cells with Vemurafenib did not effectively inhibit their ability to colonize, and cells exhibited persistent colony growth even at higher concentrations of Vemurafenib (Fig. 4B). Conversely, treatment of A375-R cells with TAK-931 demonstrated a concentration-dependent growth arrest by ~25%, when cells were treated with 50 nM of the drug. Moreover, the number of colonies dropped sharply by 80% decrease, when cells were treated with 100 nM of TAK-931 (Fig. 4C). A375-R cells treated with three different concentrations of TAK-931 in addition to 1 µM Vemurafenib (referred 10/1, 25/1 and 50/1), resulted in more effective TAK-931 concentration-dependent decrease in number of colonies compared to cells treated with individual inhibitors (Fig. 4D). Co-treatment of cells with TAK-931 (10 nM) and 1 µM Vemurafenib (10/1) caused a 32% decrease in number of colonies, while 25/1 and 50/1 combinations further reduced the number of colonies by 44% and 60%, respectively, compared to individual drug treatments as depicted in Fig. 4D. www.nature.com/scientificreports www.nature.com/scientificreports/ Protein expression of CDC7 and its correlation with clinical outcomes in melanoma tissues. To validate the in vitro studies of overexpression of CDC7 in human melanoma tissues, we performed IHC on melanoma and normal skin tissues. The immunohistochemical score of cytoplasmic CDC7 was high in 39/92 (42.4%), moderate in 43/92 (46.7%) and low in 10/92 (10.9%) of melanoma specimens. Nuclear staining was observed in 15/92 (16.3%) of melanoma tissues and in 10/10 (100%) of normal skin tissues (Fig. 5). The cytosolic staining of CDC7 in melanoma tissues was higher (p = 0.0032) compared to normal skin tissues and had a trend of significance between Stage I and III (p = 0.0763, Fig. 5E). Correlation studies showed that the cytoplasmic expression of CDC7 was significantly associated with age (r = 0.3195, p = 0.0034), gender (r = 0.2547, p = 0.0209) and pathological stage (r = 0.2810, p = 0.0167). Basically, nuclear staining of the protein was only correlated with pathological stage as shown in Supplementary Table 2.

Discussion
Although the relative success of melanoma treatment, the emergence of drug resistance still a challenge. To further study the underlying mechanisms contribute to the acquired resistance to Vemurafenib, we utilized Vemurafenib-sensitive A375 & WM983B (-P) and resistant melanoma cells A375-NRAS Q61K and WM983B-BR (-R) cells. Initially, we confirmed that the Vemurafenib resistant melanoma cells kept the acquired resistance phenotype as previously reported [21][22][23] . In cell based-assay, A375-R & WM983B-R cells treated with Vemurafenib showed a little inhibition in cellular proliferation rate, a case accompanied by consistent hyper-activation of ERK1/2 and Akt activities compared to their respective parental cells. The RAS/RAF active mutations have been detected in cutaneous melanoma and, therefore, suggesting their oncogenic activity in RAS/RAF/MEK/ERK pathway 9,15 . The gain of function of NRAS Q61K mutation constitutively hyper-activates ERK1/2 24 . Melanoma cells bearing secondary NRAS Q61K mutation are more vunerable to dvelop Vemurafenib resistance than cells with primeray BRAF mutations. This evidenced by the fact that the coexistence of NRAS Q61K and BRAF V600E in melanoma cells is sufficient to by-pass Vemurafenib inhibitory effects on ERK1/2 signaling 22 . In addition to other mechanisms, a typical mechanis of resistance is mediated by ERK1/2 hyperactivation in melanoma cells including amplification of BRAF expression, and/or mutational activation of MEK 13 . www.nature.com/scientificreports www.nature.com/scientificreports/ Our findings demonstrate that miR-3613-3p was among the most downregulated microRNAs in resistant versus parental A375-derived exosomes. However, restoration of this miR in resistant cells reversed their resistant phenotype and re-sensitized resistant melanoma cells to Vemurafenib as corroborated by our results and other previous studies conducted on resistant melanoma cells using different miRs 14,25 . Although miR-3613-3p has been reported to be dysregulated in various types of cancer, our study provides the first evidence that dysregulation of miR-3613-3p was associated with Vemurafenib resistance in melanoma cells. Prior studies elaborated on the role of miR-3613-3p in the development of drug resistance where it was downregulated in chemoresistant epithelial ovarian cancer cells to paclitaxel and carboplatin treatment 26 , and in resistant breast cancer-derived exosomes 27 .
To identify target gene candidates of miR-3613-3p, bioinformatic analyses predicted that cell division cycle 7 (CDC7) is a potential target for miR-3613-3p and, perhaps, suggesting its role in Vemurafenib resistance 28 . Our results also demonstrate that the endogenous expression of CDC7 was higher in A375-R compared to A375-P cells, whereas the candidate protein was lower or completely absent in WM983B-R cells. This also explained the reason behind downregulation of miR-3613-3p in A375-R versus WM983B-R cells corresponding to their parental control cells. CDC7 is a serine-threonine protein kinase that controls cell cycle progression through its downstream effector minichromosome maintenance 2-7 (MCM2-7), which is crucial player to unwind the double-stranded DNA as an initial step of DNA replication 29 . The DNA replication initiation machinery is a potent regulator of the proliferative state in normal cells and its dysregulation contributes to uncontrolled growth rate in cancer cells 16 . A mounting evidence indicts the overexpression of CDC7 in tumor cells and its relation with the increase in mutational frequency and therefore promotes tumorigenesis and chemoresistance 30,31 . Herein, we report that CDC7 was overexpressed in the cytoplasm of melanoma cells compared to normal skin and its expression was correlated with age, gender and tumor staging. Other previous studies also reported the overexpression of CDC7 in primary and metastatic melanoma compared to benign nevi, and was associated with lower relapse-free survival 18,32 . In the same stream, its notable expression was associated with cell cycle dysregulation, which mediates resistance to MAPK inhibitors in melanoma cells 10 .
In the present study, we found an association between lower expression of miR-3613-3p and ERK1/2 and CDC7 hyperactivation in resistant melanoma cells. Accordingly, reconstitution of miR-3613-3p in resistant A375-R cells using the miR-3613-3p mimic was associated with inhibition of ERK1/2 activity and CDC7 expression. It was also associated with a decrease in number of cell colonies compared to scrambled cells. In a recent study conducted by Li et al., the activation of ERK1/2 by EGF activates CK2α to phosphorylate PGK1 and  www.nature.com/scientificreports www.nature.com/scientificreports/ increases its interaction with CDC7 to promote DNA replication 33 . In another study, knockdown of CDC7 in oral squamous cell carcinoma reduced MCM2 and ERK1/2 phosphorylation but promoted Akt phosphorylation 34 . This suggests the link between CDC7 inhibition and reduced ERK1/2 activity. To further understand the link between CDC7 and MAPK pathway in Vemurafenib resistance melanoma, we tested the effect of Vemurafenib www.nature.com/scientificreports www.nature.com/scientificreports/ treatment on CDC7 expression in parental and resistant melanoma cells harboring BRAF V600E . Treatment of cells with Vemurafenib precluded CDC7 expression and ERK1/2 activity in parental, but not in resistant melanoma cells. This implies that CDC7 and MAPK pathway are interrelated, and CDC7 may be affected by Vemurafenib treatment in melanoma cells. However, more studies are needed to closely examine the interconnection between CDC7 and MAPK molecular components.
Inhibition of ERK1/2 activity by Vemurafenib was shown to be associated with reduced cell growth and G1-phase cell cycle arrest, which is mediated by the accumulation of CDK inhibitors 35,36 . A number of studies showed that CDKs essentially interact with CDC7 kinase to eventually activate MCM2-7 complex and promote cell proliferation 16,37 . Similar to CDKs, CDC7 may have a regulatory role on ERK1/2 activity in melanoma cells, which has not been established yet. Based on our findings, BRAF V600E inhibition blocks CDC7-downstream MCM2 and ERK1/2 activities in parental, but not in resistant cells. This was supported by the evidence that Vemurafenib treatment provoked cell cycle arrest associated with cytostatic and cytotoxic effects in parental, but not in resistant cells 38,39 . To further examine whether CDC7 inhibition could sensitize resistant melanoma cells to Vemurafenib, a pharmacologic selective CDC7 inhibitor, TAK-931, was used to manipulate the kinase activity. CDC7 inhibitors have a major potential as new generation of anti-cancer targeted therapies. Inhibition of CDC7 kinase activities resulted in cancer cell-specific apoptosis as a consequence of abrogation of cell cycle progression 30,40 . However, no previous studies have been conducted on CDC7 inhibition as an alternative targeted therapy in Vemurafenib-resistant melanoma cells. Recently, TAK-931 has been introduced as a potent selective ATP-competitive inhibitor for CDC7 kinase. TAK-931 is still under clinical trials after exerting antiproliferative effect using preclinical models. This effect was associated with a dose-dependent suppression of MCM2 phosphorylation in various solid tumors, but not in melanoma 41 . Accordingly, we established a concentration-dependent proliferation inhibition profile of Vemurafenib-sensitive and resistant melanoma cells after TAK-931 treatment. Interestingly, A375-R cells were more sensitive to TAK-931 treatment than A375-P cells. This suggests the biological role of CDC7 in developing resistance in CDC7-positive melanoma cells, and its inhibition could be a potential alternative therapy in Vemurafenib resistant melanoma cells. In contrast, WM983B-R resistant cells did not show the same effect of the drug because they do not express CDC7 as A375-R cells do. Mechanistically, we examined the effect of CDC7 inhibition by TAK-931 treatment on MAPK pathway and CDC7 downstream effector proteins, ERK1/2 and MCM-2 in A375-R cells.
A growing body of evidence shows that targeting additional signaling cascades provides additional benefit as anticancer therapy 11 . A recent study showed that mutant NRAS melanoma cells better respond to combinational therapies with better outcomes 42 . Taking the same approach, our results showed that the combination of the two drugs; Vemurafenib and TAK-931, inhibited the growth of melanoma cells compared to monotherapy. This approach may block other compensatory mechanisms involved in NRAS-dependent resistance 22 . Another advantage of using low concentrations of the two drugs is to minimize the side effects raised from the option of using monotherapy. The current strategy of melanoma treatment mainly depends on the molecular feature of tissue specimens. This study may offer a new treatment option for those patients with BRAF V600E -positive and developed resistance against Vemurafenib and have higher CDC7 expression to receive TAK-931 inhibitor in combination with the offered drug as illustrated in Fig. 6. Further studies are warranted to examine the effect of TAK-931 and Vemurafenib combination using preclinical model as a second step for reducing drug resistance in melanoma patients. In addition, melanoma specimens procured from patients with vemurafenib resistance will assist to better understand the role of CDC7 in drug resistance. Figure 6. Suggested treatment strategy in Vemurafenib-resistant melanoma patients with CDC7 inhibitor, TAK-931. Diagrammatic representation of the suggested strategy for treatment of melanoma patients with CDC7 inhibitor who had BRAF mutation and developed resistance against Vemurafenib. If CDC7 expression is high in the tissues of these patients, they are expecting to receive CDC7 specific inhibitor TAK-931 sequentially to Vemurafenib treatment and after developing signs of drug resistance. Another strategy is to increase the bioavailability of miR-3613-3p to the tumor cells, where it targets CDC7 and its downstream signaling. This intervention is anticipated to reduce the drug resistance and improve the clinical outcomes of the melanoma patients.

Materials and Methods
Cell culture. All experimental methods and protocols included in this study were performed in accordance with the National Institute of Health (NIH) and Research Compliance and Biosafety guidelines approved by Texas A&M University (IBC permit: 2016-163), College Station, Texas. Vemurafenib-resistant A375-NRAS Q61K (A375-R) and its parental A375 (A375-P) cells, established from 54-y old female malignant melanoma patient, were kindly provided by Dr. Andrew E. Aplin (Thomas Jefferson University, Philadelphia, Pennsylvania). Parental metastatic cells WM983B (WM983-P) established from inguinal node of 54-y old male and matched resistant WM983B-BR (WM983-R) cells were received from Dr. M Herlyn (Wistar Institute, Philadelphia, PA). WM983B cells harbor mutant BRAF V600E , and wild-type for NRAS, c-kit and CDK4. A375 cells were grown in DMEM (Dulbecco's Modification of Eagle's Medium) containing 4.5 g/L glucose, L-glutamine and sodium pyruvate, 10% FBS. WM983B cells were cultured in MCDB153 medium (Sigma-Aldrich, St. Louis, MO), containing 20% Leibovitz L-15 medium, 2% FBS, 5 µg/ml insulin. Cells were grown in1% penicillin/streptomycin and maintained at 37 °C in a 5% CO 2 atmosphere.
Isolation of exosomes and miR profiling. Exosomes were isolated from the conditioned media of A375-P & -R cells by ultracentrifugation as previously described 43 . RNA was extracted from the exosomes using TRIzol method following the standard protocol (ThermoFisher Scientific, Waltham, MA). The purified RNA (1 µg each) was subjected to miR profiling using miRCURY LNA ™ microRNA array kit (Qiagen, Germantown, MD), as reported 43 .
Cytotoxicity assay. About 2 × 10 3 cells were seeded in 96-well plate by adding 100 µl of the prepared cell suspension per each well and kept to settle down overnight. Next, the old media were aspired and cells were treated with different Vemurafenib (PLX4032) (Selleck Chemicals LLC, Houston, TX) concentrations ranging from 0.1 to 10 µM in triplicates, and incubated for 5 consecutive days with replenishment of the drug every 2 days. DMSO (0.1%) was used as a negative control. Cell toxicity was evaluated using cell counting kit-8 following the standard protocol (Dojindo Molecular Tech. Inc., Rockville, MD). The developed color was measured by microplate reader at 450 nm (AccuSkan FC plate reader, Thermo Fisher Scientific, Waltham, MA).
Clonogenic assay. Three hundred cells suspended in 1 ml of culture media were seeded per well in 12-well plate. Next day, cells were treated in triplicates with different concentrations of either Vemurafenib or CDC7 specific inhibitor TAK-931 (Chemietek, Indianapolis, IN), and 0.1% DMSO served as a control. Each drug was replaced with freshly prepared one every other day. After ending of incubation period, the generated colonies were thoroughly washed with PBS, and followed by 4% paraformaldehyde fixation for 15 min at room temperature. The paraformaldehyde was then aspirated and the fixed colonies were stained with 0.5% crystal violet for 25 min. Plates were then washed with water and left overnight to dry out before imaging and counting.
Transfection of melanoma cells with microRNA mimics, RNA extraction and Real-Time PCR (qPCR) analysis. Melanoma cells were cultured in 12 well-plate and about 70% confluent cells were transfected with miR-3613-3p and All Stars negative siRNA AF488 as a negative control using HiPerFect transfecting reagent (Qiagen Inc., Germantown, MD). Forty eight hours later, total RNA was extracted from melanoma cells using Trizol reagent following the manufacturer's instructions (Invitrogen Corp., Carlsbad, CA). RNA quantity and quality was measured using NanoDrop 1000. After micro-cDNA synthesis (qScript cDNA Supermix, Quanta Biosciences), qPCR was performed using CFX96 Touch TM detection system (Bio-Rad, Hercules, CA). The miRs primers miR-3613-3p and RNU-6 were purchased from Qiagen and 5 s rRNA was provided by Integrated DNA Technologies (Coralville, IA). The fold change of miR-3613-3p expression was calculated regarding U-6 and 5 s rRNA.
Western blot. Western blot analysis was performed as previously described 44 . Briefly, protein concentrations were measured by Bradford method and 30 µg protein lysate was uploaded onto a 4-20% SDS-PAGE gel (Bio-Rad, Hercules, CA) in reducing conditions. After protein fractionation step, proteins were transferred on a nitrocellulose membrane followed by blocking in 5% BSA for 1 hour. The blocked membranes were incubated overnight at 4 °C with anti-CDC7 and anti-GAPDH (Santa Cruz Biotechnology, Dallas, TX), anti-phospho-ERK (T202/Y204) and anti-ERK1/2 (Cell signaling Tech., Danvers, MA), and phospho-MCM2 and anti-MCM2 (Bethyl, Montgomery, TX) antibodies. After serial washing and incubating the membranes with the appropriate secondary antibodies for 1 hour at room temperature, the signal was developed by either Amersham ECL Prime WB Detection Reagent (GE Healthcare Life Sciences, Pittsburgh, PA) or by incubation of membranes with fluorescent secondary antibody using Odyssey CLX Imaging System (Li-COR Biosciences, Lincoln, NE).

Immunohistochemistry (IHC).
Prior of conducting any human-related studies, informed consent was obtained from all patients included in this study after IRB approval (IRB#2017-0190M) by Texas A&M University, College Station, Texas. Tissue microarray slide comprised 45 melanoma cases with 90 cores in addition to 10 normal skin cores was purchased from US Biomax, Inc. (Cat#ME1002a, Derwood, MD). The available clinical information was provided in Supplementary Table 1. IHC analysis with anti-CDC7 antibody (GeneTex Inc., Irvine, CA) was performed as we previously described 45 . Briefly, melanoma tissue sections and tissue microarray slide were de-waxed in two series of xylene and rehydrated in a descending series of ethanol. Melanoma tissue slides were then heated in 0.01 M citrate buffer (pH 6.0) for 20 min using steam cooker. The tissue sections were then immersed in 3% H 2 O 2 for 10 min to block any endogenous peroxidase activity. These sections were incubated with anti-CDC7 antibody (Santa Cruz Biotechnology, Dallas, TX) overnight at 4 °C. The antigen-antibody complex was detected by ABC Elite Kit (Vector Laboratories, Burlingame, CA) and using 3, 3′-diaminobenzidine (DAB) as chromogen. Melanoma tissue sections were counterstained with Mayer's hematoxylin solution (Newcomer