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Interferon alpha-inducible protein 6 regulates NRASQ61K-induced melanomagenesis and growth

  1. Romi Gupta
  2. Matteo Forloni
  3. Malik Bisserier
  4. Shaillay Kumar Dogra
  5. Qiaohong Yang
  6. Narendra Wajapeyee  Is a corresponding author
  1. Yale University School of Medicine, United States
  2. Agency for Science Technology and Research (A*STAR), Brenner Center for Molecular Medicine, Singapore
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Cite this article as: eLife 2016;5:e16432 doi: 10.7554/eLife.16432

Abstract

Mutations in the NRAS oncogene are present in up to 20% of melanoma. Here, we show that interferon alpha-inducible protein 6 (IFI6) is necessary for NRASQ61K-induced transformation and melanoma growth. IFI6 was transcriptionally upregulated by NRASQ61K, and knockdown of IFI6 resulted in DNA replication stress due to dysregulated DNA replication via E2F2. This stress consequentially inhibited cellular transformation and melanoma growth via senescence or apoptosis induction depending on the RB and p53 pathway status of the cells. NRAS-mutant melanoma were significantly more resistant to the cytotoxic effects of DNA replication stress-inducing drugs, and knockdown of IFI6 increased sensitivity to these drugs. Pharmacological inhibition of IFI6 expression by the MEK inhibitor trametinib, when combined with DNA replication stress-inducing drugs, blocked NRAS-mutant melanoma growth. Collectively, we demonstrate that IFI6, via E2F2 regulates DNA replication and melanoma development and growth, and this pathway can be pharmacologically targeted to inhibit NRAS-mutant melanoma.

https://doi.org/10.7554/eLife.16432.001

Introduction

Melanoma is the deadliest form of skin cancer, accounting for ~80% of skin cancer-related deaths (Miller and Mihm, 2006). Some of the most commonly observed oncogenic events in melanoma are activating mutations of the oncogenic neuroblastoma RAS viral oncogene homolog (NRAS; typically NRASQ61K/R), which occur in 20% of cases (Miller and Mihm, 2006; Tsao et al., 2012). Oncogenic NRAS causes constitutive activation of NRAS, resulting in activation of multiple downstream signaling pathways. These signaling pathways promote proliferation and reduce apoptosis to facilitate cellular transformation, tumor growth, and metastatic progression (Downward, 2003; Karnoub and Weinberg, 2008; Wellbrock et al., 2004). Key pathways regulated by NRAS include the phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase (MAPK), and Ral guanine nucleotide dissociation stimulator (RalGDS) pathways, all of which are shown to play important roles in NRAS-driven oncogenesis (Downward, 2003; Karnoub and Weinberg, 2008).

Both the PI3K and MAPK pathways have been targeted using very effective small molecule inhibitors as a clinical approach to treat RAS-mutant cancers, including NRAS-mutant melanoma. However, other pro-survival pathways can be activated by feedback and crosstalk from the pathway being targeted. For example, inhibition of the PI3K pathway alone can result in compensatory upregulation of the MAPK pathway (Carracedo et al., 2008; Serra et al., 2011). For this and several other known and unknown reasons, these approaches have not proven clinically beneficial (Baines et al., 2011). Thus, new therapeutic approaches for treating NRAS-mutant melanoma and other RAS-mutant cancers are needed.

To discover and develop more effective approaches to treat NRAS-mutant melanoma, we aimed to understand the mechanism of oncogenic NRAS-induced transformation and tumor maintenance. We identified interferon alpha-inducible protein 6 (IFI6) as a new regulator of oncogenic NRAS-induced melanocyte transformation and melanoma tumor growth, which functions by regulating E2F2 expression and consequently DNA replication. Surprisingly, we also found that NRAS-mutant melanoma cells were significantly more resistant to the drugs that induce DNA replication stress than were BRAF-mutant, NF1-deficient, or triple wild-type melanomas. In addition, treatment with the MEK inhibitor trametinib, which reduces IFI6 expression, when combined with drugs that induce DNA replication stress, potently inhibited NRAS-mutant melanoma tumor growth in cell culture and in mice. Taken together, our results identify a new role for IFI6 in E2F2-mediated regulation of DNA replication and melanoma development and growth. These studies also uncover a pharmacologically tractable DNA replication stress resistance pathway that can be targeted to inhibit NRAS-mutant melanoma.

Results

Oncogenic NRAS transcriptionally upregulates IFI6 via MAPK pathway

Oncogenic mutations in neuroblastoma RAS (NRAS), typically in codon 61, are observed in <∼20% of melanoma (2015). However, NRAS-mutant melanoma currently lacks effective targeted therapies, and targeting the pro-survival pathways downstream of oncogenic NRAS (e.g., PI3K or MEK inhibitors) have not been successful (Britten, 2013; Samatar and Poulikakos, 2014; Zhao and Adjei, 2014). Thus, a better understanding of NRAS-mutant melanoma is required for developing effective targeted therapies. Toward this end, we sought to identify factors that are necessary for oncogenic NRAS-induced melanocyte transformation and melanoma growth.

First, we performed transcriptome-wide gene expression analyses. To do so, we transformed immortalized melanocytes (MEL-ST cells) using oncogenic NRAS, NRASQ61K (hereafter referred to as MEL-ST/NRASQ61K), and then we analyzed the gene expression changes using an Illumina gene expression array. Our gene expression data analyses identified 301 genes that were significantly upregulated (p<0.05, fold-change >2.0) in MEL-ST/NRASQ61K cells compared to MEL-ST cells with an empty vector control (Supplementary file 1A and Figure 1—figure supplement 1). Among the top five genes were IL8 and IL1B, which were previously implicated in RAS-mediated transformation (Cataisson et al., 2012; Sparmann and Bar-Sagi, 2004). However, we also identified three interferon-stimulated genes (ISGs)—IFI6, IFI27, and MX1—that have not been previously implicated in oncogenic NRAS-induced transformation and melanoma tumor growth (Figure 1A and Figure 1—figure supplement 2). Furthermore, an analysis of previously published melanoma datasets revealed that IFI6 is overexpressed in melanoma samples (Figure 1B–C) (Barretina et al., 2012; Haqq et al., 2005; Riker et al., 2008; Talantov et al., 2005). Based on these results, we focused our studies on IFI6.

Figure 1 with 6 supplements see all
IFI6 is transcriptionally upregulated by NRASQ61K via MAPK pathway.

(A) Relative IFI6 mRNA expression in MEL-ST/NRASQ61K cells compared to empty vector-expressing MEL-ST cells. (B) Box plots for IFI6 mRNA expression in indicated melanoma gene expression datasets show significantly higher IFI6 mRNA expression in patient-derived cutaneous melanoma samples (2) compared to normal skin controls (1). (C) Box plot for IFI6 mRNA expression in Barretina cell line dataset. Lane 13 shows average of IFI6 mRNA expression in melanoma cell lines. (D) MEL-ST cells expressing empty vector or indicated HRAS mutants were analyzed by RT-qPCR. The relative expression of IFI6 mRNA in HRAS mutant-expressing MEL-ST cells compared to empty vector-expressing MEL-ST cells. (E) Relative expression of indicated proteins was evaluated by immunoblotting in MEL-ST cells expressing an empty vector or indicated HRAS mutants. (F) MEL-ST cells expressing empty vector or MEK-DD were analyzed for IFI6 mRNA expression by RT-qPCR. IFI6 mRNA expression in MEK-DD expressing MEL-ST cells relative to empty vector is shown. (G) MEL-ST cells expressing empty vector or MEK-DD were analyzed for indicated proteins by immunoblotting. (H) Analysis of patient-derived melanoma samples (n = 20) reveals co-expression of IFI6 with MAPK target genes. (I) Box plot for indicated melanoma gene expression dataset shows significantly higher IF6 mRNA expression in patient-derived NRAS-mutant melanoma samples (2) compared to NRAS wild-type melanoma samples (1). (J) Schematic presentation of NF-κB DNA binding site on the IFI6 promoter. (K) MEL-ST/NRASQ61K cells were analyzed for NF-κB enrichment using the ChIP assay.% NF-κB enrichment in comparison to IgG on the ACTIN or IFI6 gene promoter is shown. (L) MEL-ST cells expressing empty vector or NRASQ61K with NS or NF-κB shRNAs were analyzed for IFI6 mRNA expression by RT-qPCR. Relative IFI6 mRNA in comparison to empty vector expressing MEL-ST cells is shown. (M) MEL-ST cells expressing empty vector or NRASQ61K with NS or NF-κB shRNAs were analyzed for IFI6 protein levels by immunoblotting. ACTINB served as the loading control. In all panels, data are presented as mean ± SEM, and *p<0.05 and ***p<0.0005.

https://doi.org/10.7554/eLife.16432.002

First, we determined the mechanism by which NRASQ61K transcriptionally upregulates the expression of IFI6. Toward this end, we employed RAS mutants that specifically activate either the MAPK (HRAS v12 S35) or PI3K pathway (HRAS v12 C40). Using these mutants, we found that the MAPK pathway stimulated IFI6 expression effectively in MEL-ST cells (Figure 1D–E). To confirm this finding, we used the constitutively active MEK construct MEK-DD (Boehm et al., 2007) and found that the introduction of MEK-DD in MEL-ST cells was sufficient to stimulate IFI6 expression (Figure 1F–G). Finally, we analyzed the expression of IFI6 and key MAPK transcriptional targets in 20 patient-derived melanoma samples. We observed that IFI6 expression strongly correlated with the expression of other known MAPK transcriptional targets (Figure 1H). Additionally, IFI6 overexpression significantly correlated with the NRAS mutation status in patient-derived melanoma samples (Figure 1I) (Haqq et al., 2005). These results demonstrate that NRASQ61K activates IFI6 expression through the MAPK pathway.

In melanoma, the MAPK pathway can also be activated as a result of mutations in BRAF genes (e.g., BRAFV600E) or loss of neurofibromatosis type 1 (NF1) activity due to inactivating mutations (Coverley et al., 2002; Davies et al., 2002; Krauthammer et al., 2015). Therefore, we asked whether BRAFV600E or NF1 knockdown could result in the transcriptional upregulation of IFI6, similar to NRASQ61K. To this end, we either introduced BRAFV600E or knocked down NF1 expression in MEL-ST cells (Figure 1—figure supplement 3). As controls, we used empty vector or non-specific (NS) small hairpin RNA (shRNA), respectively. These cells were then analyzed for IFI6 expression by RT-qPCR and immunoblot analysis. Our results showed that BRAFV600E, similar to NRASQ61K, was able to activate IFI6 expression. However, NF1 knockdown did not result in IFI6 upregulation (Figure 1—figure supplement 3). These results indicate that NF1 loss is not functionally equivalent to BRAFV600E or NRASQ61K regarding its ability to activate IFI6 expression.

Next, we asked which transcription factors downstream of the MAPK pathway were necessary to activate expression of IFI6. Toward this end, we analyzed the promoter of IFI6 using rVISTA2.0 (Loots and Ovcharenko, 2004) and identified DNA binding sites for transcription factors NF-κB and STAT1 (Figure 1J and Figure 1—figure supplement 4). To test if NF-κB or STAT1 directly regulate IFI6 transcription, we first performed a chromatin immunoprecipitation (ChIP) assay. MEL-ST/NRASQ61K cells showed enrichment of NF-κB on the IFI6 promoter relative to MEL-ST cells expressing an empty vector (Figure 1K). However, we did not observe enrichment for STAT1 on the IFI6 promoter in MEL-ST/NRASQ61K cells relative to MEL-ST cells expressing an empty vector (Figure 1—figure supplement 4). To further test whether NF-κB and STAT1 influence IFI6 mRNA expression, we measured the expression of IFI6 in MEL-ST/NRASQ61K cells after knocking down the expression of either NF-κB (p65/ RelA) or STAT1. We found that NF-κB knockdown markedly decreased IFI6 expression in MEL-ST/NRASQ61K cells (Figures 1L–M and Figure 1—figure supplement 5), whereas STAT1 knockdown had no effect (Figure 1—figure supplement 5). We also determined the mechanism of NF-κB activation downstream of NRASQ61K. To this end, we knocked down the expression of IKKβ, a kinase that phosphorylates and inactivates IκB; IκB inhibits NF-κB by preventing its nuclear localization (Li et al., 1999a, 1999b). IKKβ was previously shown to be necessary for NF-κB activity and HRASv12-driven melanoma growth in mice (Yang et al., 2010). Knockdown of IKKβ resulted in reduced phosphorylation of IκB (Figure 1—figure supplement 6) and reduced NF-κB reporter activity (Figure 1—figure supplement 6). Analysis of IFI6 also revealed that IKKβ knockdown attenuated NRAS-induced IFI6 expression (Figure 1—figure supplement 6). ChIP analysis showed decreased enrichment of NF-κB on the IFI6 promoter upon IKKβ knockdown (Figure 1—figure supplement 6). Collectively, these results demonstrate that NRASQ61K, in a MAPK pathway-dependent manner via NF-κB, stimulates the transcription of IFI6.

IFI6 is required for NRASQ61K-induced transformation and NRAS-mutant melanoma tumor growth

Next, we asked if IFI6 is necessary for NRASQ61K-induced transformation. Toward this end, we knocked down the expression of IFI6 using shRNA in MEL-ST cells, and then we introduced the NRASQ61K mutant via lentiviral infection. These cells were tested for their ability to form colonies in soft agar and tumors in mice. We found that IFI6 knockdown inhibited the ability of NRASQ61K to transform MEL-ST cells, as shown by the reduced colony formation in soft agar (Figure 2A–B and Figure 2—figure supplement 1) and tumor formation in mice (Figure 2C). We also performed rescue experiments by expressing shRNA that targets the 3'-UTR of IFI6. Our results show that ectopic expression of shRNA-resistant IFI6 open reading frame (ORF) rescued growth both in soft agar and in mice (Figure 2—figure supplement 2). Notably, NF-κB knockdown also resulted in the inhibition of NRASQ61K-induced transformation, further supporting a role for NF-κB in IFI6 expression (Figure 2D–F and Figure 1—figure supplement 5).

Figure 2 with 2 supplements see all
IFI6 is necessary for NRASQ61K-induced melanocyte transformation.

(A) MEL-ST/NRASQ61K cells expressing IFI6 or non-specific (NS) shRNA were analyzed for colony-forming potential using a soft agar assay. Representative images are shown. (B) Relative colony size for the soft-agar assay presented in panel A is shown. (C) MEL-ST/NRASQ61K cells expressing IFI6 or non-specific (NS) shRNA were injected subcutaneously into the flanks of athymic nude mice. Average tumor volumes (n = 5) for MEL-ST/NRASQ61K cells expressing NS or IFI6 shRNA are shown at the indicated time points. (D). MEL-ST/NRASQ61K cells expressing NS or NF-κB shRNA were analyzed for colony-forming potential using a soft agar assay. Representative images are shown. (E) Relative colony size for the soft-agar assay presented in panel D is shown. (F) MEL-ST/NRASQ61K cells expressing NS or NF-κB shRNA were injected subcutaneously into the flanks of athymic nude mice. Average tumor volumes (n = 5) for MEL-ST/NRASQ61K cells expressing NS or NF-κB shRNA are shown at the indicated time points. In all panels, data are presented as mean ± SEM, and *p<0.05 and ***p<0.0005.

https://doi.org/10.7554/eLife.16432.009

Based on these results, we asked if IFI6 is necessary for NRAS-mutant melanoma-driven tumor growth. Toward this end, we knocked down the expression of IFI6 in three NRAS-mutant melanoma cell lines (YUGASP, M318, and SKMEL-103) and one NRAS wild-type melanoma cell line (YUVON) and monitored their colony- and tumor-forming ability. Our results show that shRNA-induced knockdown of IFI6 inhibited the ability of NRASQ61K mutant melanoma cells to form colonies in soft agar (Figures 3A–B, Figure 2—figure supplement 1 and Figure 3—figure supplement 1) and to form tumors in mice (Figure 3C). However, IFI6 knockdown did not affect colony or tumor formation in YUVON cells (Figure 3D–F). We also performed rescue experiments in YUGASP cells by expressing shRNA that targets the 3' untranslated region (UTR) of IFI6. Our results show that ectopic expression of shRNA-resistant IFI6 open reading frame (ORF) was able to rescue growth in both soft agar and in mice (Figure 3—figure supplement 2). Collectively, these results demonstrate that IFI6 is necessary for NRASQ61K-induced transformation and NRAS-mutant melanoma growth.

Figure 3 with 2 supplements see all
IFI6 is necessary for NRAS-mutant melanoma tumor growth.

(A) NRAS-mutant melanoma cell lines expressing either non-specific (NS) or IFI6 shRNA were analyzed for anchorage-independent growth using the soft agar assay. Representative images for the indicated melanoma cell lines expressing NS or IFI6 shRNA are shown. (B) Relative colony size in the soft agar assay presented in panel A are plotted for the indicated melanoma cell lines expressing either NS or IFI6 shRNA. (C) The indicated NRAS-mutant melanoma cell lines expressing either NS or IFI6 shRNA were injected subcutaneously into the flank of athymic nude mice. Average tumor volumes (n = 5) at the indicated time points are shown. (D) YUVON cells expressing NS or IFI6 shRNA were analyzed for IFI6 mRNA expression by RT-qPCR. Relative IFI6 mRNA expression (%) in YUVON cells expressing IFI6 shRNA in comparison to NS shRNA-expressing cells is shown. (E) YUVON cells expressing NS or IFI6 shRNA were analyzed for their ability to grow in soft agar. Representative wells of YUVON cells expressing the indicated shRNA and microscopic images are shown. (F) YUVON cells expressing NS or IFI6 shRNA were injected subcutaneously in athymic nude mice. Average tumor volumes (n = 5) for the indicated time points are shown. In all panels, data are presented as mean ± SEM, and *p<0.05 and **p<0.005.

https://doi.org/10.7554/eLife.16432.012

IFI6 loss results in dysregulated DNA replication via transcription factor E2F2 and inhibition of NRAS-mutant melanoma tumor growth

After confirming the role of IFI6 in the regulation of melanocyte transformation and melanoma growth, we asked how IFI6 regulates these phenotypes. To answer this question, we knocked down the expression of IFI6 in the NRAS-mutant melanoma cell line YUGASP and performed a microarray analysis (Supplementary file 1B and 1C). We found that the loss of IFI6 expression resulted in increased expression of transcription factor E2F2 and its target genes (Figure 4A and Figure 2—figure supplement 1), many of which were previously documented to regulate DNA replication (e.g., CCNE1, MCM3, MCM10) (Aladjem, 2007; Coverley et al., 2002; Thu and Bielinsky, 2013). We observed the same results in two additional NRAS-mutant melanoma cell lines, M318 and SKMEL-103 (Figure 4—figure supplement 1 and Figure 2—figure supplement 1). Furthermore, consistent with the increased level of E2F2 mRNA, we observed increased E2F2 protein level and increased E2F2 enrichment on the promoter of its target gene MCM10 (Figure 2—figure supplement 1 and Figure 4—figure supplement 2). Analysis of other E2F family genes in NRAS-mutant cells, MEL-ST/NRASQ61K cells and primary human melanocytes after IFI6 knockdown revealed no changes in the expression of other E2F genes (Figure 4—figure supplement 3). Based on these findings, we hypothesized that the loss of IFI6 results in dysregulated DNA replication via E2F2 upregulation, which in turn blocks NRAS-mutant melanoma tumor growth and melanomagenesis. To test this likelihood, we analyzed the DNA content of YUGASP cells expressing either IFI6 or NS shRNA by fluorescence-activated cell sorting (FACS). YUGASP cells expressing IFI6 shRNA displayed a significantly higher percentage of cells in S phase than the cells expressing NS shRNA (Figure 4B and Figure 4—figure supplement 4). Next, to test if IFI6 knockdown results in dysregulation of the DNA replication process, we used a DNA fiber assay to directly measure DNA replication (Merrick et al., 2004). Toward this end, we analyzed YUGASP cells expressing IFI6 or NS shRNA. Our results showed that the loss of IFI6 resulted in dysregulated DNA replication, as observed by significantly fewer ongoing DNA replication forks, significantly more stalled forks, and a significantly higher number of newly fired origins of replication compared to melanoma cells expressing NS shRNA (Figures 4C–D and Figure 4—figure supplement 4). This result also explains the high number of cells in S phase of the cell cycle.

Figure 4 with 4 supplements see all
IFI6 loss results in E2F2-mediated dysregulation of DNA replication and induction of cellular senescence in melanoma cells.

(A) YUGASP cells expressing either IFI6 or non-specific (NS) shRNA were analyzed by RT-qPCR. Expression of each gene was calculated in IFI6 knockdown cells relative to NS shRNA-expressing cells. (B) FACS analysis of YUGASP cells expressing NS or IFI6 shRNA. The percentage of cells in S phase is shown in red. (C) YUGASP cells expressing NS or IFI6 shRNA were analyzed using a DNA fiber assay. Representative images of the DNA fibers are shown. (D) YUGASP cells expressing NS or IFI6 shRNA were analyzed using the DNA fiber assay. Percentages of ongoing (ON), newly fired (NF), and terminated (Tr) DNA forks are shown. (E) YUGASP, SKMEL-103, and M318 cells expressing NS or IFI6 shRNA were analyzed for SA-β-gal activity. Representative images of cells stained for SA-β-gal activity for the indicated melanoma cell lines expressing NS or IFI6 shRNA are shown. (F) Percentage of SA-β-gal-positive cells for the experiment shown in panel E for the indicated melanoma cell lines expressing NS or IFI6 shRNA is plotted. (G) YUGASP, SKMEL-103 and M318 cells expressing each shRNA were analyzed for H3K9Ac, γH2A.X, and H2A.X using immunoblot. ACTINB was used as a loading control. In all panels, data are presented as mean ± SEM, and *p<0.05, **p<0.005.

https://doi.org/10.7554/eLife.16432.015

Previous studies have shown that dysregulated DNA replication can result in senescence induction (Bartkova et al., 2006; Di Micco et al., 2006). Therefore, we asked if IFI6 knockdown results in dysregulated DNA replication-induced cellular senescence, which may consequentially inhibit melanoma growth. To test this, we knocked down IFI6 in NRAS-mutant melanoma cell lines, YUGASP, M318, and SKMEL-103, and measured well-accepted markers of cellular senescence including senescence-associated β-gal (SA-β-gal) and acetylated histone H3K9 (H3K9Ac) (Narita et al., 2006, 2003). IFI6 knockdown resulted in the accumulation of markers of cellular senescence (Figure 4E–G), suggesting that senescence was induced. In addition, we also measured the phosphorylated histone γH2A.X as a marker of DNA damage, which showed that DNA replication stress-induced DNA damage was upregulated (Figure 4G).

Next, we asked if the E2F2 upregulation that results from IFI6 knockdown is necessary for melanoma growth inhibition. To determine this, we simultaneously knocked down the expression of E2F2 and IFI6 using shRNA (Figure 5A–B) and observed a significant rescue of the NRAS-mutant melanoma cell's ability to form colonies in soft agar (Figure 5C–D) and tumors in mice (Figure 5E–F). Furthermore, simultaneous knockdown of E2F2 and IFI6 in melanoma cells resulted in a lower percentage of cells in S phase and normalized DNA replication compared with cells expressing IFI6 shRNA (Figure 6A–B and Figure 4—figure supplement 4). We also evaluated senescence markers in this scenario and found that simultaneous knockdown of E2F2 and IFI6 prevented IFI6-induced senescence in melanoma cells (Figure 6C–E). The shRNA against E2F2 was highly specific to E2F2, as demonstrated by the unchanged mRNA levels of other E2F family genes (Figure 6—figure supplement 1).

E2F2 mediates the loss of IFI6-induced tumor suppression.

(A) YUGASP cells expressing either non-specific (NS) or IFI6 shRNA, alone or in combination with E2F2 shRNA, were analyzed for E2F2 mRNA expression by RT-qPCR. A relative E2F2 mRNA expression under the indicated conditions is shown. (B) YUGASP cells expressing either NS or IFI6 shRNA, alone or in combination with E2F2 shRNA were analyzed for E2F2, IFI6, and ACTINB protein expression by immunoblotting. (C) YUGASP cells expressing either NS or IFI6 shRNA, alone or in combination with E2F2 shRNA were analyzed for colony-forming potential. Representative soft agar images are shown. (D) Relative colony size in the soft agar assay presented in panel C under the indicated conditions is shown. (E) YUGASP cells expressing either NS or IFI6 shRNA, alone or in combination with E2F2 shRNA, were injected subcutaneously into the flank of athymic nude mice. Average tumor volumes (n = 5) formed from each cell line at the indicated times are shown. (F) Representative tumor images for the experiment presented in panel E under the indicated conditions are shown. In all panels, data are presented as mean ± SEM, and *p<0.05.

https://doi.org/10.7554/eLife.16432.020
Figure 6 with 1 supplement see all
E2F2 mediates the loss of IFI6-induced dysregulated DNA replication.

(A) YUGASP cells expressing either non-specific (NS) or IFI6 shRNA, alone or in combination with E2F2 shRNA were analyzed by FACS. The percentages of cells in each cell cycle stage are shown. (B) YUGASP cells expressing either NS or IFI6 shRNA, alone or in combination with E2F2 shRNA, were analyzed using the DNA fiber assay. The percentages of ongoing (ON), newly fired (NF), and terminated (Tr) DNA forks are shown. (C) YUGASP cells expressing either NS or IFI6 shRNA, alone or in combination with E2F2 shRNA, were analyzed for SA-β-gal activity. Representative images of cells stained for SA-β-gal activity are shown. (D) Percentage of SA-β-gal-positive cells for the experiment presented in the panel C is plotted. (E) YUGASP cells expressing each shRNA were analyzed for H3K9Ac by immunoblotting. ACTINB was used as a loading control. In all panels, data are presented as mean ± SEM, and *p<0.05, **p<0.005.

https://doi.org/10.7554/eLife.16432.021

Further studies revealed that the introduction of NRASQ61K in primary human melanocytes increased IFI6 expression and downregulated E2F2 and several E2F2 target genes, similar to melanoma cells (Figure 7A). Additionally, knockdown of IFI6 in primary human melanocytes resulted in the dysregulated DNA replication (Figure 7B and Figure 7—figure supplement 1). Furthermore, IFI6 knockdown in primary human melanocytes enhanced NRASQ61K-induced senescence, as demonstrated by markers of cellular senescence (SA-β-gal and H2K9Ac) (Figure 7C–E). Consistent with our earlier results we observed an increase in γH2A.X upon IFI6 knockdown (Figure 7E).

Figure 7 with 1 supplement see all
IFI6 loss results in DNA replication stress and senescence induction in primary human melanocytes.

(A) Primary human melanocytes expressing NRASQ61K or an empty vector were analyzed for the indicated genes by qRT-PCR. Relative gene expression is shown. (B) Primary human melanocytes expressing indicated shRNA were analyzed by DNA combing assay. Percentages of ongoing (ON), newly fired (NF), and terminated (Tr) DNA forks are shown. (C) Melanocytes expressing non-specific (NS) or IFI6, shRNA with either empty vector or NRASQ61K, were analyzed by SA-β-gal assay. Representative images are shown. (D) Percentage of SA-β-gal–positive cells expressing NS or IFI6 shRNA with either vector or NRASQ61K for the experiment presented in panel C is shown. (E) Melanocytes expressing NS or IFI6 shRNA in combination with either empty vector or NRASQ61K, were analyzed for the indicated proteins by immunoblotting. Data are presented as mean ± SEM, and *p<0.05.

https://doi.org/10.7554/eLife.16432.023

Based on these results, we tested whether immortalized melanocytes expressing NRASQ61K behaved similarly to the NRAS-mutant melanoma cells and primary human melanocytes that show regulation of E2F2 and its target genes. In complete agreement with our previous results, expression of NRASQ61K resulted in increased IFI6 expression and decreased expression of E2F2 and its target genes in MEL-ST cells (Figure 8A). We also observed dysregulated DNA replication in NRASQ61K-transformed MEL-ST cells expressing IFI6 shRNA (Figure 8B and Figure 8—figure supplement 1). However, senescence induction was not observed in MEL-ST/NRASQ61K cells expressing IFI6 shRNA (Figure 8—figure supplement 2). This is consistent with the fact that the MEL-ST cells express the SV40 early region (SV40-ER) and thus inactivate both p53 and retinoblastoma tumor suppressor (RB) pathways. However, previous studies have shown that uncontrolled DNA replication-induced DNA damage can result in apoptosis induction even in the absence of p53 and RB (Aladjem et al., 1998; Knudsen et al., 2000; Strasser et al., 1994). Therefore, we asked whether loss of IFI6 results in apoptosis induction when p53 and RB are inhibited. To this end, we tested anchorage-independent cell growth in plates coated with poly(2-hydroxyethyl methacrylate) (poly-HEMA) and using the recently developed growth in low attachment (GILA) assay (Rotem et al., 2015). We found that loss of IFI6 in MEL-ST/NRASQ61K cells increased apoptosis under anchorage-independent growth conditions compared to NS shRNA-expressing cells (Figure 8C–D). We confirmed these results by annexin V staining and immunoblot analysis of cleaved caspase 3 (Figure 8E–F). Taken together, our results demonstrate that upon p53 and RB pathway inactivation, IFI6 loss results in the upregulation of E2F2 and its target genes involved in DNA replication. This, in turn, causes DNA replication stress and consequent DNA damage, thereby inducing apoptosis. However, when the p53 and/or RB pathway is intact, IFI6 loss results in the induction of cellular senescence. This consequentially inhibits mutant NRAS-induced melanocyte transformation and melanoma growth.

Figure 8 with 2 supplements see all
IFI6 loss results in DNA replication stress and apoptosis induction in MEL-ST/NRASQ61K cells.

(A) MEL-ST cells expressing empty vector or NRASQ61K were analyzed by RT-qPCR. The expression of each gene in MEL-ST/NRASQ61K cells is shown relative to expression in empty vector control. (B) MEL-ST/NRASQ61K cells with the indicated shRNA were analyzed for DNA replication using the DNA fiber assay. The percentages of ongoing (ON), newly fired (NF), and terminated (Tr) DNA forks are shown. (C) Growth In Low Attachment (GILA) assay was performed using MEL-ST/NRASQ61K cells expressing either non-silencing (NS) or IFI6 shRNA. Cell death (%) was measured 48 hr after plating under the indicated conditions using the trypan blue exclusion assay and plotted. (D) MEL-ST/NRASQ61K cells expressing either NS or IFI6 shRNA were plated on poly-HEMA plates. Cell death (%) was measured 48 hr after plating under each condition using the trypan blue exclusion assay and plotted. (E) MEL-ST/NRASQ61K cells expressing either NS or IFI6 shRNA were plated on poly-HEMA plates. Apoptotic cell death (%) was measured 48 hr after plating under each condition by annexin V-FITC staining and plotted. (F) MEL-ST/NRASQ61K cells expressing either NS or IFI6 shRNA were plated on poly-HEMA plates. After 48 hr, the indicated proteins were analyzed by immunoblotting. ACTINB was used as a control. In all panels, data are presented as mean ± SEM, and *p<0.05.

https://doi.org/10.7554/eLife.16432.025

NRAS-mutant melanoma are resistant to DNA replication stress-inducing agents

Finally, we asked whether these results are of clinical significance. Toward this end, we tested the response of NRAS-mutant melanoma cells to drugs that induce cytotoxicity by inducing DNA replication stress. We treated NRAS-mutant (YUGASP, SKMEL-2, SKMEL-103, and M318), BRAF-mutant (SKMEL-28 and A375), NF1-mutant/null (MeWo and YUTOGS), and NRAS/BRAF/NF1 wild-type (YUVON) melanoma cells with three DNA replication stress-inducing agents: aphidicolin, camptothecin, and hydroxyurea (Durkin et al., 2008; O'Connell et al., 2010; Petermann et al., 2010). We found that NRAS-mutant melanoma cells were significantly more resistant to the cytotoxic effects of DNA replication stress-inducing drugs compared to other genotypes (BRAF-mutant, NF1-deficient, or triple wild-type) (Figure 9A). Furthermore, we noted that the knockdown of IFI6 sensitized NRAS-mutant cells to DNA replication stress-inducing agents (Figure 9B), and this effect was rescued by simultaneous knockdown of both E2F2 and IFI6 (Figure 9B). Because we had observed that the MAPK pathway regulates IFI6, we tested whether pharmacological MEK inhibitors could be combined with DNA replication stress-inducing drugs to treat NRAS-mutant melanoma. Remarkably, we found that simultaneous treatment of NRAS-mutant cells with the MEK inhibitor trametinib and either hydroxyurea or aphidicolin resulted in significantly stronger melanoma growth inhibition in cell culture (Figure 9C) and in mice (Figure 9D) than either drug alone. These results were surprising because IFI6 was also upregulated by the BRAFV600E mutation. Therefore, we asked if E2F2 and its target genes involved in the regulation of DNA replication were upregulated in BRAF-mutant, NF1-deficient, or triple wild-type melanoma cells deficient in IFI6. To do so, we knocked down the expression of IFI6 and measured the expression of E2F2 and its target genes (Figure 9—figure supplement 1). We did not observe similar changes in mRNA levels of E2F2 or its target genes after IFI6 knockdown in BRAF-mutant or NF1-deficient melanoma cell lines (Figure 9—figure supplement 1). These results indicate that only in the context of NRASQ61K, IFI6 loss results in E2F2 upregulation, which induces the expression of E2F2 target genes that regulate DNA replication. Thus, the ability of IFI6 to regulate DNA replication stress originates in its ability to repress E2F2 and its target genes specifically in the context of NRAS-mutant melanoma. Collectively, these results demonstrate that NRASQ61K upregulates IFI6, consequently repressing E2F2, thereby conferring resistance to drugs that induce cytotoxicity by causing DNA replication stress. This genetically vulnerable pathway can be targeted pharmacologically by combining a MEK inhibitor with cytotoxic agents that induce DNA replication stress to potently inhibit NRAS-mutant melanoma growth.

Figure 9 with 1 supplement see all
Targeting the DNA replication stress resistance pathway to treat NRAS-mutant melanoma.

(A) Indicated melanoma cell lines were treated with indicated drugs and analyzed by MTT assay after 48 hr of drug treatment. Survival was determined relative to vehicle-treated cells. (B) Indicated NRAS-mutant melanoma cell lines expressing IFI6 or NS shRNA, or simultaneously expressing IFI6 and E2F2 shRNA, were treated with each DNA replication stress-inducing agent for 48 hr and analyzed by MTT assay. Survival was determined relative to cells expressing NS shRNA. (C) Melanoma cells were treated with trametinib (1 nM) alone or in combination with DNA replication stress-inducing agents (0.2 µM aphidicolin or 0.25 mM hydroxyurea) for 48 hr and analyzed by MTT assay. Survival was determined relative to vehicle-treated cells. (D) Melanoma cells were injected subcutaneously into athymic nude mice (n = 5). The mice were treated on alternate days with trametinib (0.1 mg/kg, orally) alone or in combination with DNA replication stress-inducing agents (50 mg/kg aphidicolin i.p. or 50 mg/kg hydroxyurea, i.p.). Average volumes for tumors formed from each cell line at the indicated times are shown (n = 5). In all panels, data are presented as mean ± SEM, and *p<0.05.

https://doi.org/10.7554/eLife.16432.028

Discussion

In this report, we show that IFI6 regulates oncogenic NRAS-induced melanocyte transformation and NRAS-mutant melanoma tumor growth. Our findings are summarized in Figure 10 and described below. First, our results demonstrate that IFI6 expression is activated by oncogenic NRAS, which is necessary for oncogenic NRAS-induced transformation and melanoma tumor growth. Second, we report a previously undocumented role for IFI6 in E2F2-mediated regulation of DNA replication. We show that oncogenic NRAS stimulates IFI6 expression to facilitate melanocyte transformation and tumor growth. The loss of IFI6 results in E2F2-mediated dysregulation of DNA replication, resulting in cellular senescence or apoptosis and tumor growth inhibition. Finally, we show that NRAS-mutant melanomas are significantly more resistant to cytotoxic agents that function by inducing DNA replication stress than wild-type NRAS melanoma cells. These observations are highly significant because they uncover a previously undocumented genetic vulnerability that is pharmacologically amenable and can be targeted to treat NRAS-mutant melanoma. These results also have important clinical implications because there are currently no effective therapies for NRAS-mutant melanoma.

Proposed model for the role of IFI6 in melanoma tumor growth.

The model shows the mechanism by which IFI6 contributes to NRASQ61K-induced transformation and the maintenance of oncogenic NRAS-mutant melanoma tumor growth.

https://doi.org/10.7554/eLife.16432.030

IFI6 is necessary for oncogenic NRAS-induced transformation and melanoma growth

ISGs are shown to play several important biological roles. Among these, their roles in resisting and controlling pathogens are well established (Schneider et al., 2014). For example, many RNA viruses that induce the interferon response activate the expression of ISGs. This activation allows host cells to control the viral infection (Schneider et al., 2014). This phenomenon is observed in patients infected with hepatitis C virus (HCV) who receive IFN-α-based drug regimens. In patients who respond to this treatment, expression of ISGs is low in the liver before treatment and increases significantly after treatment (Sarasin-Filipowicz et al., 2008). Furthermore, increased expression of 36 unique ISGs correlated with a reduction in HCV viral load (Brodsky et al., 2007). It is important to note that some viruses, such as human cytomegalovirus (HCMV), have been shown to use specific ISGs, such as RSAD2, to enhance infection (Seo et al., 2011). We found that three ISGs—IFI6, IFI27, and MX1—were transcriptionally upregulated by oncogenic NRAS and other oncogenic RAS proteins in a MAPK pathway-dependent manner. These results show that the introduction of oncogenic RAS into human cells induces responses similar to those seen during viral infections, and oncogenic NRAS uses ISGs, such as IFI6, to facilitate cellular transformation and tumor growth.

Loss of IFI6 results in E2F2-mediated DNA replication stress

In addition to identifying the role for IFI6 in the regulation of melanoma development and tumor growth, we also discovered a previously undocumented role for IFI6 in the regulation of DNA replication. We found that the inhibition of IFI6 by shRNA-mediated knockdown results in upregulation of transcription factor E2F2, which specifically activates its target genes involved in the activation of DNA replication. This upregulation of DNA replication-promoting genes results in dysregulated DNA replication in melanocytes and melanoma cells. Some studies have shown that oncogenes induce DNA replication stress, resulting in cellular senescence (Bartkova et al., 2006; Di Micco et al., 2006), but the precise mechanism of this process was not clearly illustrated. Here, we provide evidence that loss of IFI6 results in the upregulation of E2F2, which dysregulates DNA replication. This biological process is also necessary for NRAS-induced cellular transformation and for maintaining the NRAS-mutant melanoma tumor growth. The E2F transcription factors are downstream effectors of the retinoblastoma pathway, and their transcriptional activity is required to regulate various genes, including those required for DNA replication and cell cycle progression (Bracken et al., 2004). Context-specific roles for different E2F family members have been shown, which further highlights their non-redundant biological functions (Attwooll et al., 2004). Interestingly, we found that the loss of IFI6 results in specific upregulation of E2F2 but not other E2F family genes. This observation suggests a tumor suppressor-like role for E2F2 in NRAS-mutant melanoma. Notably, although E2F family proteins are typically known to activate genes that facilitate cell cycle progression and proliferation, a number of growth inhibitory genes, such as CDKN2C (p18), CDKN2D (p19), CDKN1C (p57), E2F7, RB1 (pRB), and RBL1 are upregulated by E2F proteins during S phase (Di Stefano et al., 2003; Ortega et al., 2002; Sherr and Roberts, 1999; Stott et al., 1998). Thus, one might speculate that the induction of these growth inhibitory genes might be necessary for regulated progression of the cell cycle. For example, activation of cyclin-dependent kinase inhibitors, such as CDKN2C, CDKN2D, and CDKN1C, would hypothetically reduce CDK activity and DNA replication fork firing during late S phase. Interestingly, none of these growth inhibitory targets of E2F were upregulated after IFI6 knockdown, which may in part explain why IFI6-mediated upregulation of E2F2 results in dysregulated DNA replication and tumor growth inhibition.

Cellular senescence or apoptosis as an outcome of loss of IFI6 expression

Our study also shows that loss of IFI6 results in two different outcomes, depending on the status of the p53 and RB pathways. In melanoma cells and primary human melanocytes, IFI6 loss results in the induction of cellular senescence as a result of E2F2-mediated dysregulated DNA replication and consequent DNA damage. However, oncogenic NRAS-transformed MEL-ST cells express SV40 early region, thus inactivating both p53 and RB pathways. In this scenario, we found that the loss of IFI6 results in apoptosis induction. Our results are consistent with multiple previous studies that have demonstrated DNA damage in p53 and RB pathway-defective cells, resulting in p53- and RB-independent apoptosis (Aladjem et al., 1998; Knudsen et al., 2000; Strasser et al., 1994).

DNA replication stress resistance as a genetic vulnerability of NRAS-mutant melanoma

Our studies have identified a novel and clinically significant feature of NRAS-mutant melanoma. We found that cytotoxic agents that typically inhibit growth by inducing DNA replication stress are largely ineffective at inhibiting NRAS-mutant melanomas. In contrast, BRAF-mutant, NF1-deficient, and triple wild-type melanomas were relatively sensitive to these agents. We also confirm resistance to these agents is due in part to the oncogenic NRAS-mediated upregulation of IFI6 and consequential repression of E2F2 and its target genes. Knockdown of IFI6 increased the sensitivity of NRAS-mutant melanoma to DNA replication stress-inducing agents, and simultaneous loss of E2F2 and IFI6 rescues this effect. Notably, knockdown of IFI6 in BRAF-mutant, NF1-deficient, or triple wild-type melanoma did not alter the expression of E2F2 or its target genes. This in part explains the differences in the sensitivity of melanoma genotypes to agents that induce cytotoxicity through DNA replication stress. Because IFI6 is upregulated in NRAS-mutant melanoma, and its expression correlates with NRAS mutation status, this subgroup of melanoma could be targeted using this therapeutic approach. Therefore, the DNA replication stress pathway represents a unique vulnerability of NRAS-mutant melanoma that can be pharmacologically inhibited to achieve tumor inhibition in cell culture and in vivo. Several of the agents we used here, such as aphidicolin, camptothecin, and hydroxyurea, are used in clinical cancer treatment (Geyer and Mesa, 2014; Patel et al., 2006; Sargent et al., 1996). Based on our results, we speculate that these drugs will have limited utility in patients harboring NRAS and potentially other RAS mutations. Thus, our results may have clinical application for the identification of patient populations that might benefit from these drugs.

Materials and methods

Cell culture and plasmids

SKMEL-2, SKMEL-28, MeWo, and A375 cells were purchased from American Type Culture Collection (ATCC) and grown as recommended. Neonatal primary human melanocytes were purchased from Life Technologies and grown as recommended by the supplier. All short-term melanoma cultures (YUGASP, YUHEF, YUTOGS, and YUVON) were obtained from the Yale SPORE in Skin Cancer, Yale University, and grown as recommended. SKMEL-103 and M318 cells were provided by Dr. Keiran Smally (Moffitt Cancer Center, Florida). MEL-ST cells were provided by Prof. Robert Weinberg (Whitehead Institute, MIT). All the cell lines were authenticated using STR analysis and tested for mycoplasma regularly using a MycoAlert Mycoplasma detection kit (Lonza, Allendale, NJ). Human IFI6 open reading frame was cloned in pcDNA3.1/hygro and used for rescue experiments. The plasmids pBabe puro-HRAS V12 (plasmid #15269), pBabe puro-HRAS V12 S35 (plasmid# 12274), pBabe puro-MEK-DD (plasmid #15268), and pBabe-puro-BRAFV600E (plasmid #15269) were purchased from Addgene. FG12 was a kind gift of Prof. David Baltimore, and FG12/NRASQ61K was a kind gift of Maria Soengas (CNIO, Spain).

Microarray experiments and data analysis

For microarray experiments using MEL-ST cells, total RNA was isolated from MEL-ST cells transduced by FG12-NRASQ61K or vector control and used to generate labeled antisense RNA. For microarray experiments using YUGASP cells, total RNA was isolated from YUGASP cells expressing either non-specific (NS) or one of two different IFI6 shRNA sequences and used to generate labeled antisense RNA. All antisense RNAs were made using the Ambion MessageAmp Kit and hybridized to Illumina HumanHT-12 V4.0 expression BeadChip using Illumina’s protocol.

The microarray data were processed using GenomeStudio (Illumina), log2-transformed and quantile-normalized using the 'lumi' package of Bioconductor. All samples passed quality-control (QC) assessment, which included checking various control plots as suggested by Illumina, as well as other standard microarray-related analyses. Differential expression analyses were performed using the 'limma' package, and a moderated t-test with a Benjamini-Hochberg multiple testing correction procedure was used to determine statistical significance (adjusted p-value, <0.05). Pathway analysis of differentially expressed genes for each comparison was performed using MetaCore (version 6.8 build 29806; GeneGo). Microarray data were submitted to Gene Expression Omnibus (Accession No. GSE39294 for MEL-ST cell experiments, and GSE69933 for YUGASP experiments).

Melanoma dataset analysis

The Talantov melanoma dataset (Talantov et al., 2005), Haqq melanoma dataset (Haqq et al., 2005), Barretina cell line dataset (Barretina et al., 2012), and Riker melanoma dataset (Riker et al., 2008) were downloaded from Oncomine (https://www.oncomine.org) and analyzed for IFI6 expression and plotted as box plots between melanoma and normal skin samples. The Talantov melanoma dataset analyzed 45 cutaneous melanomas, 18 benign melanocytic skin nevi, and 7 normal skin samples using Affymetrix U133A microarrays. The Haqq melanoma dataset analyzed 25 melanomas, 9 non-neoplastic nevi, and 3 normal skin samples using a cDNA microarray. The Cancer Cell Line Encyclopedia (CCLE) project (i.e., Barretina cell line dataset) is a compilation of gene expression data from 917 human cancer cell lines comprising 18 different tumor types. Lane 13 represents 57 melanoma cell lines. The Riker melanoma dataset analyzed 40 metastatic melanomas, 42 primary skin cancers, 4 normal skin samples, and 1 normal skin primary cell culture using Affymetrix HG U133 Plus 2.0 microarrays.

RNA preparation, cDNA preparation, and quantitative PCR analysis

For mRNA expression analyses, total RNA was extracted with TRIzol (Invitrogen) and purified using RNeasy mini columns (Qiagen). The cDNA was generated using the M-MuLV first-strand cDNA synthesis kit (New England Biolabs) according to the manufacturer’s instructions. Quantitative RT-PCR was performed using the Power SYBR Green kit (Applied Biosystems) according to the manufacturer’s instructions. Actin was used as an internal control. Primer sequences are provided in Supplementary file 1D.

Chromatin immunoprecipitation

The IFI6 promoter sequence was downloaded from the UCSC genome browser and analyzed using rVista 2.0. ChIP experiments were performed as described previously (Gazin et al., 2007). Normalized Ct (ΔCt) values were calculated by subtracting the Ct obtained with input DNA from that obtained with immunoprecipitated DNA [ΔCt = Ct(IP) − Ct(input)]. Relative fold enrichment of a factor at the target site was then calculated using the formula 2−[ΔCt(T) − ΔCt(Actb)], where ΔCt(T) and ΔCt(Actb) are ΔCt values obtained using target and Actb (irrelevant) primers, respectively.

shRNA, retrovirus, and lentivirus preparation

IFI6, E2F2, STAT1, and RelA (the p65 subunit of NF-κB), NF1 shRNA sequences in either pLKO.1 or pZIPZ lentiviral expression vectors were obtained from Open Biosystems. For retrovirus or lentivirus production viral expression, constructs, and viral packaging plasmids were co-transfected into 293T cells using Effectene (Qiagen), following the manufacturer’s recommendations. Supernatant fractions were collected 48 hr after the transfection, and purified virus particles were used to infect primary or melanoma cell lines and were selected by growth on puromycin or by sorting GFP-positive cells using a flow cytometer.

Antibodies and immunoblot analysis

Whole cell protein extracts were prepared using IP lysis buffer (Pierce) containing Protease Inhibitor Cocktail (Roche) and Phosphatase Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO). Protein concentration was estimated using a Bradford Assay kit (Bio-Rad). Proteins were resolved on 10% or 12% polyacrylamide gels and transferred to PVDF membranes using a wet transfer apparatus from Bio-Rad. Membranes were blocked with 5% skim milk and probed with primary antibodies, followed by the appropriate secondary HRP-conjugated antibody (GE Healthcare, UK). Blots were developed using the SuperSignal Pico Reagent (Pierce). Information about the antibodies used in this study is provided in Supplementary file 1D.

Melanoma sample analysis

Total RNA from melanoma samples were obtained from the University of Massachusetts Medical School (UMMS) Tissue and Tumor Bank and analyzed for the expression of IFI6 and MAPK target genes FOSL1, ETV5, SPRY2, and DUSP6 by RT-qPCR.

Soft agar assay and tumorigenesis assay

For the soft agar assay, individual cell lines were seeded in triplicate at three different dilutions, ranging from 5 × 103 to 2 × 104 cells. Cells were seeded into a 0.4% soft agar layer. After 4 weeks, colonies were stained with 0.005% crystal violet solution and counted. Each experiment was repeated at least twice. Athymic nude (NCr nu/nu) mice (aged six weeks) were injected subcutaneously with cell lines either expressing different shRNAs or transduced with the empty vector. Tumor volume was calculated using the formula: length × width2 × 0.5. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at Yale University. For mouse experiments trametinib was dissolved in 0.5% hydroxypropyl methylcellulose and was administered orally (0.1 mg/kg) every other day. Hydroxyurea and aphidicolin were dissolved in sesame oil and administered by intraperitoneal (i.p.) injection every other day (50 mg/kg) alone or in combination with trametinib (0.1 mg/kg).

DNA fiber assay

The DNA fiber assay was performed as described previously (Merrick et al., 2004). Briefly, cells were plated in the appropriate medium until they reached 30–40% confluency. After 48 hr, IdU (Sigma-Aldrich: I7125) was added to the exponentially growing cells (final concentration: 25 µM), and the cells were incubated for 30 min at 37°C in 5% CO2. After washing with PBS, cells were incubated with a second label, CldU (Sigma-Aldrich: C6891), at the final concentration of 250 µM for additional 30 min at 37°C. Cells were trypsinized and counted, and 2 × 103 cells in 3 µl of medium were used for each slide. The 3 µl of cell suspension was applied to the end of the glass slide and air-dried for 5 min. Cells were lysed by adding 7 µl of lysis solution (50 mM EDTA and 0.5% SDA in 200 mM Tris-HCl, pH 7.6). Glass slides were placed at a 15º angle to allow the DNA fibers to spread across the length of the slide, and then were placed horizontally to air dry. After this, slides were fixed with methanol:acetic acid (3:1) for 10 min, washed with double distilled water, and treated with 2.5 M HCl for 30 min. Next, the fixed cells were blocked with 5% BSA for 30 min at room temperature and incubated with primary antibodies (anti-BrdU [mouse antibody], BD Biosciences #347580 for IdU at a 1:25 dilution and anti-BrdU [rat antibody], Abcam # ab6326 for CldU at a 1:400 dilution, each in 5% BSA) for 1 hr at room temperature in a humidified chamber. Slides were then washed three times with 1× PBS for 5 min and then incubated with secondary antibodies (1:500 sheep anti-mouse Cy3, Sigma, Cat# C218-M for IdU, and 1:400 goat anti-rat Alexa Fluor 488, Invitrogen, cat A11006 for CldU) in 5% BSA for 1 hr at room temperature in the dark. After secondary antibody incubation, glass slides were washed and visualized at 63X magnification to locate the fibers. Pictures were captured with one color channel, and data were analyzed with image analysis software ImageJ.

Senescence-induced beta-galactosidase (SA-β-gal) staining

The SA-β-gal assay was performed as described previously (Dimri et al., 1995). Briefly, 2.0 × 105 cells were plated in 6-well plates and stained after 48 hr. First, the cells were washed twice with 1X PBS and fixed with 4% paraformaldehyde for 5 min at 37°C. Then the cells were washed twice more with 1X PBS and incubated with β-gal staining solution (made fresh in 1× PBS: X-gal 1 mg/ml (0.1%), potassium ferricyanide 5 mM, potassium ferrocyanide 5 mM, MgCl2 2 mM, NaCl 150 mM, and citric acid/sodium phosphate solution 40 mM) in a 37°C incubator in the dark. The reaction was terminated by removing the staining solution and washing the cells twice with distilled water. Cells were visualized with an inverted bright-field microscope, and the images were captured using the 10× objective. The percentage of SA-β-gal–positive cells was plotted with respect to the total number of cells used in each case.

Fluorescence-activated cell sorting (FACS)

FACS analyses were conducted as described previously (Santra et al., 2009). Briefly, cells were fixed with 70% ethanol overnight. The following day, cells were washed twice with 1× PBS and suspended in 300 µl of 1× PBS, treated with RNase (Sigma-Aldrich) and propidium iodide for 1 hr, and analyzed using FACSCalibur (BD Biosciences).

MTT assay

For this assay, 5 × 103 cells were plated in a 100 µl volume in 96-well plates. After 48 hr, inhibitors (i.e., aphidicolin, camptothecin, hydroxyurea, and trametinib), used at a range of concentrations was mixed in 100 µl of medium and added to the cells. After 48 hr of inhibitor treatment, the cell viability was evaluated. To do this, 20 µl of 5 mg/ml MTT solution dissolved in 1× PBS was added to each well and incubated for 1 hr at 37°C incubator. The MTT solution was removed gently, and 100 µl of DMSO were added. After mixing well by pipetting, absorbance was measured at 590 and 630 nm. An average was calculated for both readings, and then measurement at 630 nm was subtracted from that at 590 nm. The relative growth rate was plotted with respect to vehicle-treated control cells.

Luciferase reporter assay

MEL-ST/NRASQ61K cells expressing IFI6 shRNA were transfected with a NF-κB-firefly luciferase construct (Promega) along with a control TK-Renilla luciferase plasmid (pRL-TK). After 24 hr, the cells were lysed in passive lysis buffer, and the luciferase reporter assay was performed using the Dual-Luciferase Reporter Assay Kit (Promega). Relative NF-κB activity was measured as the ratio of firefly to Renilla luciferase activity and reported as the average of triplicate measurements.

Poly-HEMA assay and growth in low attachment (GILA) assay

To evaluate anoikis, 6-well tissue culture plates were coated with 200 μL poly-HEMA (Sigma) and left for 10 hr in a laminar flow hood. MEL-ST/NRASQ61K cells expressing NS or IFI6 shRNA were seeded in the poly-HEMA-coated plates and incubated for 48 hr at 37°C. The cells were then counted using trypan-blue exclusion assay by a hemocytometer. The experiments were performed in triplicate.

GILA assay was performed as described previously (Rotem et al., 2015). Briefly, 25,000 MEL-ST/NRASQ61K cells expressing NS or IFI6 shRNA were seeded in 12-well ultra low-attachment plates (Corning) and incubated for 48 hr at 37°C. The cells were then counted using trypan-blue exclusion assay by a hemocytometer. The experiments were performed in triplicate.

Apoptosis measurement using annexin V/ propidium iodide staining and cleaved caspase 3 immunoblot

MEL-ST/NRASQ61K cells expressing NS or IFI6 shRNA were analyzed for apoptosis by flow cytometry using the FITC-Annexin Apoptosis Detection Kit I (BD Pharmingen), as per the manufacturer’s protocol. Caspase 3 cleavage was detected by immunoblotting using an anti-cleaved caspase 3 antibody (Cell Signaling Technology).

Statistical analysis

All the experiments were conducted in three biological replicates. The results for individual experiments were expressed as mean ± SEM. The P-values were calculated by t-test using GraphPad Prism version 6.0 hr for Macintosh, GraphPad Software, San Diego, California, USA (www.graphpad.com).

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Decision letter

  1. Joaquín M Espinosa
    Reviewing Editor; University of Colorado School of Medicine, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Interferon Αlpha-Inducible Protein 6 regulates NRASQ61K-induced melanomagenesis and growth by regulating DNA replication" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Zhi Sheng (Reviewer #2) and a member of our Board of Reviewing Editors, and the evaluation has been overseen by Kevin Struhl as the Senior Editor.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. The required experiments represent a substantial investment and it will be good to hear back from you with a plan and an estimate of the time it may take to complete the required additions and changes.

The current manuscript by Gupta et al. describes the putative involvement of the interferon-stimulated gene IFI6 in the development of NRAS mutated melanoma. The authors describe a molecular pathway whereby mutations in the NRAS oncogene lead to upregulation of IFI6 expression via the activation of MEK kinases and the NF-κB transcription factor. Since IFI6 depletion leads to deregulated DNA replication and senescence, the authors propose a model whereby IFI6 upregulation downstream of NRAS is required for cell proliferation and tumor growth.

The reviewers and Senior Editor discussed the manuscript at length and after much deliberation reached a consensus, deciding to invite a resubmission of a thoroughly revised manuscript addressing the points below. A key point discussed by the reviewers is the fact that the mechanism illuminated is based on cell-based work and xenograft studies, and that a key test of the conclusions would involve the use of genetically-engineered mouse (GEM) models. However, the reviewers agreed that requiring the use of GEM models would be outside of the scope of the current manuscript, and that a thorough response to the concerns raised below could be satisfactory.

The major concerns are:

1) Specificity for NRAS-driven tumors. The manuscript describes a mechanism that is supposedly specific for NRAS-driven melanomas, but reviewers pointed out that the involvement of the RAF->MEK->ERK pathway indicates that melanomas driven by mutations in BRAF or silencing of NF1 should also display elevated IFI6 mRNA expression. This point should be carefully investigated and clarified. Is IFI6 upregulation unique to NRAS-driven tumors or not? Furthermore, by what mechanism(s) does NRAS-activated RAF->MEK->ERK activate NF-κB? Is it a direct mechanism of activation or does this occur through the secretion of inflammatory cytokines leading to NF-κB activation? If NRAS activates NF-κB signaling through the RAF->MEK->ERK pathway as described, then why wouldn't BRAF(V600E) or NF1(Null) melanomas, which have readily detectable pERK1/2 signaling, not also activate IFI6 mRNA expression? Indeed, it was noted that in their "NRAS wild-type" melanoma cell lines the authors employed at least one cell line (A375), which expresses BRAF(V600E) and has readily detectable pERK1/2 signaling. In sum, additional experiments are required to provide mechanistic insight into the mechanism of NRAS-mediated IFI6 expression through NF-κB activation and thus clarify the source of specificity (or lack thereof) for NRAS-driven melanoma.

2) Deregulated DNA replication versus senescence. The authors conclude that RNAi-mediated silencing of IFI6 leads to senescence, but this is not well documented and somewhat contradictory. The cells display elevated levels of SA- -β-gal, a widely used but unreliable marker of senescence, and a decrease in H3K9 acetylation. However, the cells also display increased numbers of cells in S phase, which is contrary to the idea of senescence, an irreversible form of cell cycle arrest generally associated with arrest in G1. How can these observations be reconciled? Could the authors detect evidence of DNA damage such as collapsed DNA replication forks, gH2AX foci and/or the recruitment of RAD51 or 53BP1 into damage sites?

Furthermore, in their initial gene induction studies and throughout the manuscript that authors employed Mel-ST cells, which are melanocytes immortalized by expression of hTERT and rendered susceptible to oncogenic transformation by expression of the SV40 Large and small T/t antigens. Since Large T binds to and inhibits the activity of the pRB, p107 and p130 "pocket proteins" that are negative regulators of E2F activity, and also to TP53, it is hard to understand mechanistically how senescence could be elicited in cells that are lacking these key tumor suppressors that are known to enforce the senescence program. Moreover, the authors claim that shIFI6 leads to elevated E2F2 mRNA expression (Figure 3A) but since, to all intents and purposes, Mel-ST cells are TP53, pRB, p107 and p130 deficient, the significance of this observation is questionable since these cells should already have abundant E2F activity. It is also notable that the authors have not assessed expression of E2F2 protein or the DNA binding/transcriptional activity of E2F2 in the cells under study. The authors claim that their data "demonstrates that IFI6 is a new regulator of DNA replication". Of note, IFI6 knockout mice are normal and cells engineered to over-express do not show any obvious alterations in DNA synthesis. It is not clear where IFI6 is located in the cell or whether it is a part of the DNA replication machinery. Hence, there are no hints from previous publications, nor any compelling evidence from this manuscript, that "IFI6 is a new regulator of DNA replication". In sum, additional experiments would be needed to clarify the relationship between deregulated IFI6 expression, E2F control, accelerated DNA replication and the establishment of senescence.

3) Correlation between sensitivity to DNA replication stress-inducing drugs and pERK levels. The relative sensitivity of NRAS mutated melanoma cells to the cytotoxic effects of DNA replication stress-inducing drugs should be assessed compared to the levels of pERK1/2 signaling and IFI6 expression in the control (NRAS wild-type) cells. Since A375 cells express BRAF(V600E), the differences in sensitivity may not be related to pERK1/2 levels. This should be further investigated and clarified.

4) Lack of correlation between NRAS mutation status and IFI6 expression in the TCGA dataset. The authors conclude that mutational activation of NRAS in melanocytes drives expression of the known interferon-regulated genes IFI6, IFITM1 and MX1. However, this conclusion is not supported by analysis of TCGA data for cutaneous melanoma that comprises ~478 well curated specimens of which 278 specimens are informative for NRAS, BRAF or NF1 mutation and the expression of IFI6, IFITM1 and MX1 mRNAs. Using the MSKCC cBio Portal (http://www.cbioportal.org/public-portal/), one of the Reviewers interrogated this dataset and found no correlation between mutational activation of NRAS and the expression of the IFI6, IFITM1 and MX1 mRNAs. Indeed, mutational activation of NRAS and the expression of these mRNAs tends towards mutual exclusivity. The authors should address this concern, and the revised manuscript should discuss analysis of public datasets, and to what extent these analyses support (or do not) the overall molecular mechanism described here.

5) Missing experimental controls. The authors fail to provide an immunoblot that documents diminished IFI6 expression in the knockdown cells. Reviewers request a rescue experiment in which an RNAi resistant form of IFI6 rescues the knockdown phenotype with regard to colony formation in agarose, and if possible, tumorigenicity in mice. The authors fail to present an immunoblot that documents diminished E2F2 expression in the shE2F2 knockdown cells. Have the authors conducted specificity experiments to confirm that their siRNAs are working through E2F2? How was NF-κB knocked down in Figure 1G? Please include an immunoblot that demonstrates knockdown of a component of NF-κB.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Interferon Α-Inducible Protein 6 regulates NRASQ61K-induced melanomagenesis and growth by regulating DNA replication" for further consideration at eLife. Your revised article has been favorably evaluated by Kevin Struhl as the Senior editor and a Reviewing editor.

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

Given the concerns raised by the reviewers about the indirect role of IFI6 in control of DNA replication, it is proposed that the title of the manuscript be changed from:

'Interferon Αlpha-Inducible Protein 6 regulates NRASQ61K-induced melanomagenesis and growth by regulating DNA replication'

to:

‘Interferon Αlpha-Inducible Protein 6 regulates NRASQ61K-induced melanomagenesis and growth’

Similarly, in the Abstract, the sentence:

'Collectively, we demonstrate that IFI6 via transcription factor E2F2 regulates DNA replication and melanoma development and growth and this pathway can be pharmacologically targeted to inhibit NRAS mutant melanoma.'

could be changed to:

'Collectively, we demonstrate that IFI6, via E2F2, regulates DNA replication and melanoma development and growth, and this pathway can be pharmacologically targeted to inhibit NRAS mutant melanoma.'

In the Introduction, the sentence:

'In addition, pharmacological inhibition of IFI6 with the MEK inhibitor trametinib, when combined with drugs that induce DNA replication stress'

should be changed to:

'In addition, treatment with the MEK inhibitor trametinib, which reduces IFI6 expression, when combined with drugs that induce DNA replication stress'

Results section, paragraph four, it should refer to Figure 2—figure supplement 2, not Figure 2—figure supplement 1. Authors make consider switching the order of these two figure supplements, so that they flow better with the text.

https://doi.org/10.7554/eLife.16432.037

Author response

The major concerns are:

1) Specificity for NRAS-driven tumors. The manuscript describes a mechanism that is supposedly specific for NRAS-driven melanomas, but Reviewers pointed out that the involvement of the RAF->MEK->ERK pathway indicates that melanomas driven by mutations in BRAF or silencing of NF1 should also display elevated IFI6 mRNA expression. This point should be carefully investigated and clarified. Is IFI6 upregulation unique to NRAS-driven tumors or not? Furthermore, by what mechanism(s) does NRAS-activated RAF->MEK->ERK activate NF-κB? Is it a direct mechanism of activation or does this occur through the secretion of inflammatory cytokines leading to NF-κB activation? If NRAS activates NF-κB signaling through the RAF->MEK->ERK pathway as described, then why wouldn't BRAF(V600E) or NF1(Null) melanomas, which have readily detectable pERK1/2 signaling, not also activate IFI6 mRNA expression? Indeed, it was noted that in their "NRAS wild-type" melanoma cell lines the authors employed at least one cell line (A375), which expresses BRAF(V600E) and has readily detectable pERK1/2 signaling. In sum, additional experiments are required to provide mechanistic insight into the mechanism of NRAS-mediated IFI6 expression through NF-κB activation and thus clarify the source of specificity (or lack thereof) for NRAS-driven melanoma.

We thank the reviewers for asking these important questions. To address these concerns thoroughly, we performed several new experiments.

First, we showed that ectopic expression of BRAFV600E in MEL-ST cells, similar to NRASQ61K, results in the upregulation of IFI6 expression, which is consistent with the role of the MAPK pathway in the upregulation of IFI6. These new results are presented in Figure 1—figure supplement 3 of this revised manuscript.

Second, we knocked down the expression of NF1 in MEL-ST cells but did not observe an upregulation of IFI6. This indicates that there are still unknown effects of NF1 loss beyond MAPK pathway activation involved in the regulation of IFI6 expression. These results also show that NF1-deficient and NRAS-mutant melanoma are not equivalent, because NRASQ61K was able to activate IFI6, but loss of NF1 was not (also see our response to point 4 in this regard). These new results are presented in Figure 1—figure supplement 3.

The regulation of NF-κB by RAS proteins is a well-studied phenomenon. Therefore, to determine the mechanism of NF-κB upregulation, we first surveyed the literature and found that IKKβ is necessary for NF-κB activation in a mouse model of HRASv12-driven melanoma and for HRASv12-driven melanoma growth (see Yang et al., 2010, JCI PMID: 20530876). This study also showed that HRASv12 activates IKKβ kinase activity to activate NF-κB. IKKβ activates NF-κB via inflammatory cytokines, and in absence of IKKβ, inflammatory cytokines cannot activate NF-κB expression (see Li et al., 1999, J Exp. Med. PMID: 10359587 and Li et al., 1999, Science PMID: 10195897).

Therefore, we knocked down IKKβ expression in MEL-ST cells expressing NRASQ61K and asked if this inhibits NF-κB activity and the ability of NRASQ61K to activate IFI6. Similar to the results reported by Yang et al., we found that knockdown of IKKβ prevented NF-κB activation, as assessed by decreased IκBα phosphorylation, decreased NF-κB-responsive luciferase reporter activity, and decreased binding of NF-κB to the IFI6 promoter and the inability of NRASQ61K to activate IFI6 expression. These new results are presented in Figure 1—figure supplement 6 of this revised manuscript.

2) Deregulated DNA replication versus senescence. The authors conclude that RNAi-mediated silencing of IFI6 leads to senescence, but this is not well documented and somewhat contradictory. The cells display elevated levels of SA-β-gal, a widely used but unreliable marker of senescence, and a decrease in H3K9 acetylation. However, the cells also display increased numbers of cells in S phase, which is contrary to the idea of senescence, an irreversible form of cell cycle arrest generally associated with arrest in G1. How can these observations be reconciled? Could the authors detect evidence of DNA damage such as collapsed DNA replication forks, gH2AX foci and/or the recruitment of RAD51 or 53BP1 into damage sites? Furthermore, in their initial gene induction studies and throughout the manuscript that authors employed Mel-ST cells, which are melanocytes immortalized by expression of hTERT and rendered susceptible to oncogenic transformation by expression of the SV40 Large and small T/t antigens. Since Large T binds to and inhibits the activity of the pRB, p107 and p130 "pocket proteins" that are negative regulators of E2F activity, and also to TP53, it is hard to understand mechanistically how senescence could be elicited in cells that are lacking these key tumor suppressors that are known to enforce the senescence program. Moreover, the authors claim that shIFI6 leads to elevated E2F2 mRNA expression (Figure 3A) but since, to all intents and purposes, Mel-ST cells are TP53, pRB, p107 and p130 deficient, the significance of this observation is questionable since these cells should already have abundant E2F activity. It is also notable that the authors have not assessed expression of E2F2 protein or the DNA binding/transcriptional activity of E2F2 in the cells under study. The authors claim that their data "demonstrates that IFI6 is a new regulator of DNA replication". Of note, IFI6 knockout mice are normal and cells engineered to over-express do not show any obvious alterations in DNA synthesis. It is not clear where IFI6 is located in the cell or whether it is a part of the DNA replication machinery. Hence, there are no hints from previous publications, nor any compelling evidence from this manuscript, that "IFI6 is a new regulator of DNA replication". In sum, additional experiments would be needed to clarify the relationship between deregulated IFI6 expression, E2F control, accelerated DNA replication and the establishment of senescence.

The second set of concerns also included several important questions. First, the reviewers mentioned that we did not document the senescence phenotypes very well. As the reviewers noticed, we used two established criteria (SA-β-gal assay and H3K9 acetylation) used by others to identify senescent cells. The use of H3K9Ac measurement was based on previous studies demonstrating increased heterochromatinization in senescent cells (for an example, see Narita et al., 2006, Cell PMID: 16901784).

Therefore, together, these markers are predictive of cellular senescence. As an additional DNA damage marker, we have now included p-H2A.X immunoblot. Regarding the collapsed DNA replication fork, in the previous version of our manuscript, we used the gold standard method for measuring DNA replication stress: the DNA combing assay. This assay clearly showed dysregulation of DNA replication, and, in conjunction with p- H2A.X, demonstrates DNA damage. These new results are presented in Figure 4G, Figure 7E, and Figure 8F of this revised manuscript.

The statement that senescence results in G1 phase cell cycle arrest is not entirely accurate, because the state of cell cycle arrest depends upon various factors including cell type and stimuli that induce senescence. For example, in cellular senescence, cells can be arrested in the G2/M phase (for a review on this topic, please refer to Gire and Dulic, 2015, Cell Cycle PMID: 25564883).

Second, regarding the statement that an increase of cells in S phase is counterintuitive for a senescence phenotype, we find that the loss of IFI6 results in E2F2-mediated unscheduled DNA replication (as demonstrated in the DNA combing assay, as well as the activation of several genes involved in DNA replication). As a result, cells incorporate more nucleotides, which causes a surge of cells in the S phase. Once this uncontrolled replication reaches a threshold level, the cell can no longer divide and therefore undergoes senescence. Please note that two previous studies have also documented this phenomenon in the context of cellular senescence. We cited these studies in our previous submission (Bartkova et al., 2006, Nature PMID: 17136093 and Di Micco et al., 2006 Nature PMID: 17136094) However, we understand the confusion this might have created for the reviewers; therefore, we have clearly articulated these results in the revised manuscript and added a paragraph in the Discussion section to clarify this point.

Third, we agree with the reviewers regarding the question of senescence induction in MEL-ST/NRASQ61K cells upon IFI6 knockdown. In the previous version of the manuscript, we did not test MEL-ST/NRASQ61K cells expressing IFI6 shRNA for cellular senescence induction, but tested only melanoma cell lines and primary human melanocytes. Therefore, we tested cellular senescence induction in MEL-ST cells transformed with NRASQ61K and expressing either IFI6 shRNA or nonspecific shRNA as a control. As suggested by the reviewers, we did not observe senescence in MEL-ST/NRASQ61K cells expressing IFI6 shRNA (see Figure 8—figure supplement 2). This is consistent with published studies demonstrating that the p53 and RB pathways are required for senescence induction.

We, then, asked what could be a possible reason for the attenuation of NRASQ61K-induced transformation upon IFI6 knockdown in MEL-ST/NRASQ61K cells. Previous studies have shown that uncontrolled DNA replication and consequent DNA damage can result in apoptosis induction in a p53- or RB-independent manner (for examples, see Strasser et al., 1994, PMID: 7954799; Knudsen et al., 2000, PMID: 11003670; and Aladjem et al., 1998, PMID: 9443911). To determine whether increased apoptosis induction might explain the observed phenotype in MEL-ST cells, we measured cell death and apoptosis using various assays (poly-HEMA coated plates, growth in detached condition assay (GILA), annexin V-FITC staining, and cleaved caspase 3 by immunoblotting). These assays were performed under detached conditions that mimic soft agar and in vivo tumor formation. We found that loss of IFI6 increased apoptosis induction in MEL-ST/NRASQ61K cells. These new results are presented in the Figure 8 of this revised manuscript. Taken together, these results show that loss of IFI6 expression results in DNA replication stress-induced DNA damage, which can cause senescence or apoptosis depending upon the presence or absence of active p53/RB proteins.

Finally, in regard to E2F gene activity/function, it is important not to generalize the functions of all E2F proteins. Previous studies have shown significant differences in these proteins, and context-specific functions for several of them have been described (for examples, see Attwooll et al., 2004, PMID: 15538380; DeGregori et al., 1997, PMID: 9207076; and DeGregori, 2002, Biochim Biophys Acta PMID: 12020800). It is also important to note that oncogenes can enhance the expression and/or activity of E2F family proteins (for an example, see Berkovich and Ginsberg, 2001, JBC PMID: 11551910).

However, we agree with the reviewers that it is important to show that E2F2 expression and function are enhanced after IFI6 knockdown. Therefore, we measured both RNA and protein level of E2F2 and our result showed that loss of IFI6 does result in increased E2F2 mRNA and protein expression (Figure 2—figure supplement 1). To show increased enrichment of E2F2 on the promoters of DNA replication regulatory genes that are upregulated in IFI6 knockdown cells, we performed ChIP experiments. Our results show increased enrichment of E2F2 on the MCM10 promoter after IFI6 knockdown. These new results are presented in Figure 4—figure supplement 2 of this revised manuscript.

In regard to the IFI6 knockout mice, we are not sure which study the reviewers are referring to. We do not find any evidence of IFI6 knockout mice in the literature. Moreover, none of our studies were performed using mouse cells, so we cannot comment on the role of IFI6 in mouse cells.

Regarding overexpression studies, we are not aware of any study that contradicts our results. Moreover, the fact that there is no hint of alteration in DNA replication in previous studies does not mean much unless a DNA replication stress phenotype was actively investigated. Moreover, none of the previous studies were performed in melanocytes or melanoma cells; therefore, it is hard to draw conclusions or determine the relevance of those previous studies in relation to our study.

Regarding, IFI6 being a regulator of DNA replication, we did not state that IFI6 directly regulates DNA replication by associating with the DNA replication machinery. Instead we showed that IFI6 loss results in the upregulation of E2F2, which in turn activates the expression of genes that are known regulators of DNA replication. Therefore, IFI6 loss results in uncontrolled DNA replication via upregulation of E2F2 and its target genes that regulate DNA replication, which is a more accurate description of our results and was discussed in our previous manuscript. Dysregulated DNA replication is one of the outcomes of IFI6 loss. Based on the questions from the reviewers, we have modified the language of our manuscript to more clearly show that the effect of IFI6 on DNA replication is not direct but rather mediated through E2F2.

3) Correlation between sensitivity to DNA replication stress-inducing drugs and pERK levels. The relative sensitivity of NRAS mutated melanoma cells to the cytotoxic effects of DNA replication stress-inducing drugs should be assessed compared to the levels of pERK1/2 signaling and IFI6 expression in the control (NRAS wild-type) cells. Since A375 cells express BRAF(V600E), the differences in sensitivity may not be related to pERK1/2 levels. This should be further investigated and clarified.

The above-mentioned point concerns the specificity of resistance towards the DNA replication stress-inducing agents. To further expand our results and determine the reason for this drug resistance, we performed additional experiments using additional BRAF-mutant and NF1-deficient melanoma cell lines. Our results reconfirmed our findings that NRAS-mutant melanoma cell lines were far more resistant to DNA replication stress-inducing drugs than other melanoma genotypes. These new results are presented in Figure 9A of this revised manuscript.

To determine whether IFI6 is upregulated by both oncogenic NRAS and oncogenic BRAF, and why only NRAS-mutant cell lines are resistant to DNA replication stress-inducing agents, we tested if the same set of genes is upregulated upon IFI6 knockdown in BRAF-mutant, NF1-deficient, and triple wild-type melanoma cells. We found that IFI6 knockdown did not result in increased expression of E2F2 or target genes that regulate DNA replication in BRAF-mutant, NF1-deficient, or triple wild-type melanoma cells, as was observed in NRAS-mutant melanoma cells. Thus, the sensitivity of melanoma cells to DNA replication stress-inducing drugs is not due to the ability of a genetic alteration (such as BRAFV600E) to simply activate IFI6 alone. Rather, it depends upon the ability of IFI6 to regulate DNA replication stress through E2F2-mediated transcription of key DNA replication target genes, which is seen only in the context of NRAS-mutant melanoma. These new results are presented in the Figure 9—figure supplement 1 of this revised manuscript.

4) Lack of correlation between NRAS mutation status and IFI6 expression in the TCGA dataset. The authors conclude that mutational activation of NRAS in melanocytes drives expression of the known interferon-regulated genes IFI6, IFITM1 and MX1. However, this conclusion is not supported by analysis of TCGA data for cutaneous melanoma that comprises ~478 well curated specimens of which 278 specimens are informative for NRAS, BRAF or NF1 mutation and the expression of IFI6, IFITM1 and MX1 mRNAs. Using the MSKCC cBio Portal (http://www.cbioportal.org/public-portal/), one of the Reviewers interrogated this dataset and found no correlation between mutational activation of NRAS and the expression of the IFI6, IFITM1 and MX1 mRNAs. Indeed, mutational activation of NRAS and the expression of these mRNAs tends towards mutual exclusivity. The authors should address this concern, and the revised manuscript should discuss analysis of public datasets, and to what extent these analyses support (or do not) the overall molecular mechanism described here.

Before describing our findings using the TCGA dataset, we would like to point out that in our previous submission, we presented results from several publically available datasets, in which we found that IFI6 expression was upregulated in melanoma samples compared to normal skin samples (see Figure 1B,C and I). We also showed an analysis from a study that confirmed that IFI6 expression correlates with NRAS mutation status in clinical melanoma samples (see Figure 1I).

We would also like to point out that based on the analysis of TCGA dataset there is no tendency for NRAS-mutant melanoma and expression of IFI6, IFIMT1, and MX1 to be mutually exclusive, because the p-values for these comparisons do not reach significance (p-value for IFI6 comparison in NRAS-mutant melanoma=0.555, p-value for IFI27 comparison in NRAS-mutant melanoma = 0.391, and p-value for MX1 comparison in NRAS-mutant melanoma =0.226).

Finally, and most importantly, we believe that the statement above assumes that the TCGA melanoma dataset is powerful enough to identify all MAPK target genes or BRAF/NRAS/NF1 signature genes. Thus, we put the TCGA melanoma dataset to this test. To this end, we selected two previously published studies and analyzed 71 genes in total. The first study identified 43 BRAF-MAPK-regulated target genes using BRAFV600E and MEK inhibitors (Joseph et al., 2010, PNAS PMID: 20668238), and the second study presented a large-scale analysis of patient-derived cutaneous melanoma samples that identified 29 genes that predict a mutant BRAF signature in melanoma (Kannengiesser et al., 2008 Molecular Oncology PMID: 19383316). Of the 43 genes from the Joseph et al. study, only 7 showed a significant tendency towards co-occurrence with the BRAFV600E mutation, and 5 showed a significant tendency towards mutual exclusivity with BRAFV600E. Surprisingly 31 of the 43 genes showed no correlation with the BRAFV600E mutation in the TCGA dataset.

Because in principal, as mentioned by the reviewers, oncogenic NRASQ61K and inactivation of NF1 should activate MAPK targets and correlate with these genotypes, we analyzed all 43 genes in the context of NRAS mutation or NF1 mutation/deletions. Our results showed that only 3 of the 43 genes showed a significant tendency towards co- occurrence in NRAS-mutant melanoma sample and 3 of the 43 genes showed a significant tendency towards mutual exclusivity, whereas 37 showed no correlation. Notably, these genes expect one did not show the same tendencies between the BRAF- and NRAS-mutant melanoma samples. In NF1-mutant melanomas, 3 of these 43 genes showed a significant tendency toward co-occurrence, and none of these 3 genes were common with NRAS-mutant and BRAF-mutant melanoma samples. Overall, this analysis showed that the TCGA data analysis identified less than 30% of MAPK targets in melanoma and less than 15% MAPK targets in NRAS- and less than 10% in NF1-mutant samples. Thus lack of correlation in the TCGA data between IFI6 expression and NRAS mutation should not be considered surprising based on this analysis.

The study by Kannengiesser et al. identified 29 genes that are upregulated in BRAF-mutant melanoma patient samples and thus represent a BRAF-mutant melanoma signature. Similar to our analysis with the Joseph et al. study, only 6 of the 2930 genes showed a significant tendency towards co-occurrence with BRAF-mutant melanoma. Thus, it failed to identify 23 of the 30 29 (796%) BRAF signature genes reported by Kannengiesser et al. Furthermore, only 1 gene showed a significant tendency towards co- occurrence with NRAS-mutant melanoma, and 7 showed a significant tendency toward mutual exclusivity. Finally, only 2 gene showed a significant tendency towards co- occurrence with NF1-deficient melanoma, and only one of these genes were identified to be significant for NRAS-mutant melanoma but not for BRAF-mutant melanoma.

Based on these preliminary analyses of the TCGA dataset we identified several genes that correlated with NRAS or NF1 mutations, but not with both genotypes, indicating differences between NRAS-mutant and NF1-deficient melanomas. This also provides a partial explanation for the differential regulation of IFI6 under NRAS-mutant and NF1-deficient conditions.

Thus, we conclude that datasets such as TCGA, although useful tools for researchers, are not comprehensive enough to predict or identify all biological outcomes in any given tumor type. We believe that the lack of correlation between IFI6 expression and NRAS mutation represents a limitation of the TCGA dataset rather than the proof that it is not regulated by the MAPK pathway. Therefore, independent studies, such as ours, should be encouraged and are complementary to the existing literature and large- scale datasets, including TCGA. We have included this analysis for the reviewers for review purpose as Supplementary file 2 and would be happy to include it as part of our manuscript if the reviewers recommend that we do so.

5) Missing experimental controls. The authors fail to provide an immunoblot that documents diminished IFI6 expression in the knockdown cells. Reviewers request a rescue experiment in which an RNAi resistant form of IFI6 rescues the knockdown phenotype with regard to colony formation in agarose, and if possible, tumorigenicity in mice. The authors fail to present an immunoblot that documents diminished E2F2 expression in the shE2F2 knockdown cells. Have the authors conducted specificity experiments to confirm that their siRNAs are working through E2F2? How was NF-κB knocked down in Figure 1G? Please include an immunoblot that demonstrates knockdown of a component of NF-κB.

As per the reviewers’ request, we included immunoblots that are in agreement with loss of IFI6 mRNA, confirming that IFI6 protein was also efficiently reduced in cells expressing IFI6 shRNA. These results are presented in Figure 2—figure supplement 1.

To show the specificity of the phenotype, we performed rescue experiments using two representative cell lines from our studies (YUGASP and MEL-ST/NRASQ61K). Our results show that ectopic expression of the IFI6 open reading frame (ORF) rescues growth in the soft agar assay and tumor growth in mice caused by IFI6 shRNA targeting the 3'-UTR. These new results are presented in Figure 2—figure supplement 2 and Figure 3—figure supplement 2.

We included data showing that E2F2 shRNA knocks down both E2F2 mRNA and protein levels. These new results are presented in Figure 5A and B.

We also show that IFI6 loss activates E2F2 but not other E2F family genes. These new results are presented in Figure 2—figure supplement 1 and Figure 4—figure supplement 3. Furthermore, we show that E2F2 knockdown does not affect the expression of other E2F family genes, thus E2F2 shRNA is highly specific to E2F2. These new results are presented in Figure 6—figure supplement 1.

Regarding the NF-κB blot, NF-κB was knocked down by shRNA that targets p65 (RelA). We included an immunoblot to show that we were also able to knock down p65 at both the mRNA and protein levels. These new results are included in Figure 1—figure supplement 5.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Given the concerns raised by the reviewers about the indirect role of IFI6 in control of DNA replication, it is proposed that the title of the manuscript be changed from:

'Interferon Α-Inducible Protein 6 regulates NRASQ61K-induced melanomagenesis and growth by regulating DNA replication'

to:

Interferon Α-Inducible Protein 6 regulates NRASQ61K-induced melanomagenesis and growth

As suggested, we have changed the title to the recommended title.

Similarly, in the Abstract, the sentence:

'Collectively, we demonstrate that IFI6 via transcription factor E2F2 regulates DNA replication and melanoma development and growth and this pathway can be pharmacologically targeted to inhibit NRAS mutant melanoma.'

could be changed to:

'Collectively, we demonstrate that IFI6, via E2F2, regulates DNA replication and melanoma development and growth, and this pathway can be pharmacologically targeted to inhibit NRAS mutant melanoma.'

As suggested, we have changed the above-mentioned sentence to as recommended by the reviewers.

In the Introduction, the sentence:

'In addition, pharmacological inhibition of IFI6 with the MEK inhibitor trametinib,when combined with drugs that induce DNA replication stress'

should be changed to:

'In addition, treatment with the MEK inhibitor trametinib, which reduces IFI6 expression, when combined with drugs that induce DNA replication stress'

We have now modified the recommended sentence in Introduction to the sentence recommended by the reviewers.

Results section, paragraph four, it should refer to Figure 2—figure supplement 2, not Figure 2—figure supplement 1. Authors make consider switching the order of these two figure supplements, so that they flow better with the text.

As the reviewers may have noticed that the Figure 2—figure supplement 1 shows immunoblotting results for IFI6 knockdown and E2F2 expression in multiple cell lines. Because we wanted to test if IFI6 is required for NRASQ61K-induced transformation, we generated IFI6 knockdown in NRASQ61K transformed MEL-ST. Figure 2—figure supplement 1 shows effective IFI6 knockdown at protein level in this cell line. Please note that results illustrating reduced colony formation in soft agar after IFI6 knockdown is shown in Figure 2A–B. Additionally, Figure 2—figure supplement 2 shows our results with the ectopic expression of shRNA-resistant IFI6 open reading frame (ORF) in NRASQ61K transformed MEL-ST expressing IFI6 shRNA. Therefore, there is no change required for the figure citations.

https://doi.org/10.7554/eLife.16432.038

Article and author information

Author details

  1. Romi Gupta

    Department of Pathology, Yale University School of Medicine, New Haven, United States
    Contribution
    RG, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  2. Matteo Forloni

    Department of Pathology, Yale University School of Medicine, New Haven, United States
    Contribution
    MF, Conception and design, Acquisition of data, Analysis and interpretation of data
    Competing interests
    The authors declare that no competing interests exist.
  3. Malik Bisserier

    Department of Pathology, Yale University School of Medicine, New Haven, United States
    Contribution
    MB, Conception and design, Acquisition of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  4. Shaillay Kumar Dogra

    Singapore Institute of Clinical Sciences, Agency for Science Technology and Research (A*STAR), Brenner Center for Molecular Medicine, Singapore, Singapore
    Contribution
    SKD, Performed the analysis and interpretation of all microarray data presented in this manuscript and worked for GEO submission of the files
    Competing interests
    The authors declare that no competing interests exist.
  5. Qiaohong Yang

    Department of Pathology, Yale University School of Medicine, New Haven, United States
    Contribution
    QY, Helped with the acquisition of data presented in the Figure 2 and Figure 3-figure supplements
    Competing interests
    The authors declare that no competing interests exist.
  6. Narendra Wajapeyee

    Department of Pathology, Yale University School of Medicine, New Haven, United States
    Contribution
    NW, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    For correspondence
    narendra.wajapeyee@yale.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3306-349X

Funding

National Institutes of Health (R01CA200919)

  • Narendra Wajapeyee

National Institutes of Health (R21CA195077-01A1)

  • Narendra Wajapeyee

National Institutes of Health (R21CA191364-01)

  • Narendra Wajapeyee

National Institutes of Health (R21CA197758-01)

  • Narendra Wajapeyee

Melanoma Research Alliance (Pilot grant award)

  • Narendra Wajapeyee

National Institutes of Health (R01CA196566)

  • Narendra Wajapeyee

National Institutes of Health (R21CA197758)

  • Narendra Wajapeyee

American Cancer Society (128347-RSG-15-212-01-TBG)

  • Narendra Wajapeyee

Melanoma Research Alliance

  • Narendra Wajapeyee

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Prof. Joann Sweasy, Yale University, for help with the DNA combing assay. We gratefully acknowledge grants from the National Institutes of Health: R01CA200919 (NW), R01CA196566 (NW), R21CA197758 (NW), R21CA191364 (NW), R21CA195077 (NW), and NW is also supported by a Research Scholar Grant from the American Cancer Society (128347-RSG-15-212-01-TBG) and grants from the Melanoma Research Alliance.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols of the Yale University (IACUC protocol #2016-11333).

Reviewing Editor

  1. Joaquín M Espinosa, University of Colorado School of Medicine, United States

Publication history

  1. Received: March 28, 2016
  2. Accepted: September 7, 2016
  3. Accepted Manuscript published: September 8, 2016 (version 1)
  4. Version of Record published: September 21, 2016 (version 2)

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© 2016, Gupta et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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