PTENP1-AS contributes to BRAF inhibitor resistance and is associated with adverse clinical outcome in stage III melanoma

BRAF inhibitors (BRAFi) selectively target oncogenic BRAFV600E/K and are effective in 80% of advanced cutaneous malignant melanoma cases carrying the V600 mutation. However, the development of drug resistance limits their clinical efficacy. Better characterization of the underlying molecular processes is needed to further improve treatments. We previously demonstrated that transcription of PTEN is negatively regulated by the PTEN pseudogene antisense RNA, PTENP1-AS, and here we investigated the impact of this transcript on clinical outcome and BRAFi resistance in melanoma. We observed that increased expression levels of PTENP1-AS in BRAFi resistant cells associated with enrichment of EZH2 and H3K27me3 at the PTEN promoter, consequently reducing the expression levels of PTEN. Further, we showed that targeting of the PTENP1-AS transcript sensitized resistant cells to BRAFi treatment and that high expression of PTENP1-AS in stage III melanoma correlated with poor survival. Collectively, the data presented here show that PTENP1-AS is a promising target for re-sensitizing cells to BRAFi and also a possible prognostic marker for clinical outcome in stage III melanoma.

Although cutaneous malignant melanoma is a molecularly diverse disease, approximately 50% carry activating mutations in the serine/threonine protein kinase BRAF. The majority of BRAF mutations are represented by a valine (V) to glutamic acid (E) or lysine (K) substitution at position 600 (BRAF V600E/K ) 1 . These missense mutations occur in the BRAF kinase domain, which results in constitutively active BRAF and subsequently activated MAPK signaling. Drug development efforts using a targeted approach have successfully led to the development of small inhibitory molecules (BRAFi, i.e. vemurafenib, dabrafenib, encorafenib), which specifically target the oncogenic BRAF V600E/K . While 80% of patients with advanced melanoma carrying a BRAF V600E/K mutation initially respond well to this treatment, resistance emerges within a median of 6-7 months of treatment 2 . Although the time to emergence of resistance can be prolonged to 11 months when combined with MEK inhibitors, the 5-year overall survival rate is only 34% for the combined treatment 3 . In recent years there has been a growing understanding of the underlying molecular mechanisms involved in acquired BRAFi resistance 4,5 and activation of the PI3K/AKT pathway due to loss of PTEN has been found to contribute to this process 6 . Therefore, better molecular understanding of the mechanisms that contribute to suppression of PTEN is desired and might help to identify novel approaches that could circumvent or delay the onset of resistance.
Approximately 14,000 pseudogenes have been identified in the human genome and roughly 900 of these have been reported to be transcribed into non-coding RNAs (ncRNAs) 7 . Molecular functions have been characterized for only a handful of these pseudogenes [8][9][10][11] , for instance through microRNA (miRNA) sponging 10  www.nature.com/scientificreports/ acting antisense RNAs (asRNA) 8 . One of these, the PTEN pseudogene, PTENP1 (also known as PTENpg1 and PTENΨ), is divergently transcribed into sense (PTENP1-S) and asRNA (PTENP1-AS) transcripts. While PTENP1-S promote expression of PTEN by functioning as a miRNA sponge for PTEN related miRNAs 10 , PTENP1-AS acts as a transcriptional suppressor by inducing epigenetic alterations at the PTEN promoter 8,12 . Interestingly, loss of PTEN expression has been found during the development of resistance to BRAFi 6 and melanoma metastasis 13,14 , but the involvement of PTENP1-AS remains unknown. In this study, our aim was to characterize the impact of PTENP1-AS on clinical outcome in stage III melanoma and BRAFi treatment efficacy. We observe that the expression of PTENP1-AS is induced in BRAFi resistant sublines in relation to the parental sensitive cell line. This coincides with transcriptional suppression of PTEN, likely through the recruitment of EZH2 and subsequent formation of H3K27me3 at the PTEN promoter. Finally, we also demonstrate that expression of PTENP1-AS is increased in tumor samples from stage III melanoma patients with poor survival. In summary, our study provides insights into the involvement of PTENP1-AS in resistance to BRAFi treatment.

Results
Inverse correlation of PTEN and PTENP1-AS in BRAFi resistant A375 sublines. Previous studies suggested downregulation of PTEN to be important upon the development of resistance to BRAFi 6,13 . Based on these reports, we set out to investigate if PTENP1-AS was involved in this process through transcriptional suppression of PTEN. To do this, a series of BRAFi resistant melanoma sublines were obtained by culturing the BRAFi sensitive A375 cell line in increasing doses of BRAFi (A375PR1 (resistant to PLX4720), A375VR3 and A375VR4 (both resistant to vemurafenib)) 15 . Compared to the parental A375 cells, IC 50 measurements for the A375PR1 and A375VR4 cell lines confirmed roughly six-to eight-fold increased tolerance to vemurafenib (Supplementary Fig. S1), thus in agreement with a previous study 15 . We next measured PTEN expression using Western blot and qRTPCR and observed downregulation of PTEN at the protein (Fig. 1a, Supplementary Fig. S2a) as well as mRNA (Fig. 1b, Supplementary Fig. S2b) levels. Importantly, the negatively regulated downstream target of PTEN, p-AKT, showed a corresponding increase in phosphorylation compared to the parental A375 cell line (Fig. 1a, Supplementary Fig. S2a).
We next measured levels of PTENP1-encoded transcripts and identified a significant upregulation of PTENP1-AS in all three resistant sublines (Fig. 1c, Supplementary Fig. S2c) and also a modest, although non-significant, induction of PTENP1-S (Fig. 1d, Supplementary Fig. S2d). Since previous studies had identified multiple isoforms of PTENP1-AS with distinct regulatory functions 8 , we carefully investigated different isoforms in one of the sublines (A375PR1). Multiple primer sets for the different variants of PTENP1-AS were assessed on semi-qRTPCR and revealed increased expression of all isoforms (Fig. 1e, Supplementary Fig. S3). Notably, unspliced RNA levels of PTENP1-AS was also found to be induced, indicating that PTENP1-AS is activated at the transcriptional level. Collectively, these initial observations motivated us to further elucidate the role of PTENP1-AS in resistance to BRAFi.

PTENP1-AS suppresses PTEN in BRAFi resistant cells through chromatin remodeling.
To investigate the molecular interplay between PTENP1-AS and PTEN in greater detail, we focused, from here, on the A375PR1 subline. We designed a gapmer antisense oligonucleotide (ASO) (Fig. 2a,b) and also an siRNA targeting the PTENP1-AS transcript (Fig. 2c,d) and confirmed knockdown in A375 as well as in A375PR1 cells using qRTPCR. Intriguingly, knockdown of PTENP1-AS only induced the expression of PTEN in the A375PR1 cells, while PTEN remained unaffected in the vemurafenib sensitive A375 cells ( Fig. 2a-d), suggesting that PTENP1-AS is predominantly active in the resistant subline. In contrast, no effect on PTENP1-S expression levels was observed in either cell line upon knockdown of PTENP1-AS ( Supplementary Fig. S4).
We next asked if suppression of PTEN in the A375R1 subline was due to epigenetic changes at the PTEN promoter since high promoter methylation has been commonly observed in melanoma and is associated with poor clinical outcome 16 . PTEN promoter CpG methylation was assessed by the methyl-cytosine-specific McrBc restriction enzyme. qRTPCR showed increased enzymatic cleavage in the A375PR1 subline, compared to the parental A375 cells (Fig. 3a), thus suggesting increased PTEN promoter CpG methylation in the resistant subline. Since we have previously reported PTENP1-AS to modulate transcription of PTEN through the recruitment of EZH2 and DNMT3A 8,12 , we next measured levels of EZH2 and H3K27me3 at the PTEN promoter. Chromatin immunoprecipitation (ChIP) followed by qRTPCR revealed increased levels of EZH2 and H3K27me3 at the PTEN promoter in the A375PR1 subline (Fig. 3b,c), while the baseline expression levels of DNMT3A and EZH2 were similar in the resistant and parental cell lines as measured by qRTPCR (Fig. 3d,e). To further interrogate their role in suppression of PTEN, EZH2 and DNMT3A were depleted using siRNAs. Although individual siRNA-mediated knockdown only resulted in a modest induction of PTEN (Fig. 3f,g, Supplementary Fig. S5a,b), simultaneous knockdown generated over a twofold induction of PTEN in the A375PR1 cells, in contrast to the parental A375 cells, where the expression of PTEN was largely unchanged (Fig. 3h, Supplementary Fig. S5c,d). Altogether, these data suggest that suppression of PTEN is mediated by PTENP1-AS likely through the recruitment of EZH2 by a mechanism that may be exclusively active in the vemurafenib resistant cells.

C/EBPB is a transcriptional regulator of PTENP1-AS.
To explore mechanisms underlying transcriptional induction of PTENP1-AS (Fig. 1c), we took advantage of FANTOM5 CAGE data and ChIP-sequencing data from the UCSC Genome Browser and identified a binding site for the transcription factor (TF) C/EBPB close to the PTENP1-AS transcriptional start site (TSS) (Fig. 4a). Comparing A375PR1 to A375 cells, revealed an increased RNA expression of C/EBPB by qRTPCR (Fig. 4b), and ChIP further identified increased levels of C/EBPB at the PTENP1-AS promoter (Fig. 4c)  www.nature.com/scientificreports/ expression of unspliced PTENP1-AS in the A375PR1 cells (Fig. 4d), while no significant change was identified for PTENP1-S ( Supplementary Fig. S6), thus suggesting that C/EBPB functions as a transcriptional activator of PTENP1-AS.

PTENP1-AS is a clinically relevant target and a prognostic marker in melanoma.
We next explored if the resistant A375PR1 cells could be sensitized to BRAFi by targeting the PTENP1-AS transcript. Gapmer ASO-mediated suppression did not have an effect on the basal levels of cell death but sensitized resistant cells to treatment with 10 μM of vemurafenib ( Fig. 5a), as measured by annexin V and propidium iodide staining. To further validate this effect, we performed a colony formation assay and also included a second resistant subline, A375VR4. While gapmer ASO-mediated knockdown of PTENP1-AS had minor effect on colony formation in parental A375, both resistant sublines were clearly sensitized to vemurafenib, in particular at lower concentrations (1 μM) (Fig. 5b, Supplementary Fig. S7). We finally set out to investigate the relevance of PTENP1-AS in tumors from melanoma patients. The expression levels of PTEN and PTENP1-AS were first assessed in melanoma samples from the TCGA (The Cancer Genome Atlas program) where although weak but a significant negative correlation was observed (r = − 0.20, p = 1.6e −3 , Fig. 5c). The expression of PTENP1-AS was next determined in lymph node metastases from a cohort of 20 stage III melanoma patients. The samples were initially chosen to include two groups of patients, with either long (≥ 60 months) or short (≤ 13 months) overall survival 17 , and the expression of PTENP1-AS was determined by qRTPCR. Notably, 9/10 (90%) patients having tumors with high expression of PTENP1-AS (median cut-off) www.nature.com/scientificreports/ had poor survival, while low expression of PTENP1-AS seemed to be indicative of prolonged survival, although not reaching significance (p = 0.06) (Fig. 5d). This motivated us to evaluate the expression of PTENP1-AS in an independent second cohort of stage III melanoma metastasis samples. Lymph node metastases from 29 stage III melanoma patients were analyzed for the expression of PTENP1-AS. The patient samples from the second cohort were divided in high (n = 12) or low (n = 17) expression of PTENP1-AS, as determined by qRTPCR, and a survival plot was generated (Fig. 5e). A significant difference in overall survival was observed between patients with high and low expression of PTENP1-AS. In summary, the expression of PTENP1-AS appears to be a promising prognostic marker for clinical outcome, where increased expression of PTENP1-AS correlates with poor overall survival.

Discussion
The data presented in this study suggest a role for PTENP1-AS in the development of resistance to BRAFi. On the basis of our data, we suggest that expression of PTENP1-AS is induced by the TF C/EBPB in BRAFi resistant melanoma cell lines, which results in transcriptional suppression of PTEN through the recruitment of EZH2 with subsequent formation of H3K27me3 (Fig. 3b,c) and DNA methylation of the PTEN promoter (Fig. 3a). Moreover, we find that expression of PTENP1-AS predicts clinical outcome in stage III melanoma patients, www.nature.com/scientificreports/ where high expression of PTENP1-AS in first regional lymph node metastases correlates with poor overall survival. In summary, our findings bring important information about molecular functions of PTENP1-AS upon development of BRAFi resistance and better understanding of this pathway could reveal novel approaches to re-sensitize drug resistant cells.
Our data are consistent with our previous findings demonstrating that PTENP1-AS is a negative regulator of PTEN through chromatin remodeling of the PTEN promoter 8 . Targeting the PTENP1-AS transcript using gapmer ASOs and siRNAs generate a moderate induction of PTEN exclusively in the resistant subline (Fig. 2), thus demonstrating that the PTENP1-AS transcript does not act as an on/off switch for PTEN expression, but rather causes subtle variations of transcription in a phenotype-dependent manner 8 . These variations of PTEN expression are within physiological relevant levels where a modest decrease of 20% have been shown to increase cancer susceptibility 18 .
Our data is also consistent with previous RNA-sequencing studies showing induction of C/EBPB in BRAFi resistant cells 19 . However, these previous studies did not report PTEN or PTENP1-AS as candidate genes involved in BRAFi resistance, possibly due to the applied thresholds since the cut-off was set to a fold change ≥ 2. Based www.nature.com/scientificreports/ on our study, both PTEN and PTEN1-AS are likely to be excluded from such an analysis when these thresholds are applied. Several PTENP1-encoded lncRNAs with opposing functions have been described. While the miRNA sponge model suggests concordant expression of PTEN and PTENP1-S 10 , we have not observed this phenomenon in the present study, despite a modest induction of PTENP1-S (Fig. 1d, Supplementary Fig. S2d). Instead, the PTENP1-AS transcript appears to be the dominant regulator of PTEN expression under these conditions. However, this does not exclude that miRNA sponging could take place as well. A regulatory mechanism, where translation as well as transcription of PTEN is regulated through lncRNAs encoded by the PTENP1 locus is still a plausible scenario. Subtle variations of PTEN expression have previously been reported to be associated with cancer susceptibility 18 , suggesting that strict and highly ordered regulation of PTEN expression is crucial for evading   Expression levels of PTENP1-AS was measured in first regional lymph node metastases from an independent set of 29 stage III melanoma patients using qRTPCR. The p-value represents a log-rank test. www.nature.com/scientificreports/ carcinogenesis. It is therefore likely that PTEN is regulated at several different levels. Deletions of PTENP1 have been reported in melanoma cell lines and tissues, supporting that suppression of PTEN also occurs at the posttranscriptional level through miRNA-mediated regulation 20 . It is also likely that tissue and cell type specific expression levels of i.e. DNMT3A, EZH2 and miRNAs dictate the regulatory outcome of PTENP1-encoded lncRNAs, and PTENP1-S may also be involved in sponging of miRNAs related to other mRNAs beyond PTEN 21 .
Notably, this study also shows that the suppression of PTEN in BRAFi resistant cells may be reversible through targeting of EZH2, DNMT3A as well as PTENP1-AS. The individual knockdown of EZH2 or DNMT3A did not re-activate the expression of PTEN to the same extent as a simultaneous knockdown of these proteins (Fig. 3f-h). We speculate that this is due to incomplete knockdown of DNMT3A and EZH2 where some active protein complexes may still be present under these circumstances (Supplementary Fig. S5). An interaction between EZH2 and DNMT3A have been demonstrated 22 and the combined knockdown of EZH2 and DNMT3A may in this case be more efficient in reducing these protein complexes. Importantly, the induction of PTEN is more prominent in the A375PR1 cells than in the parental cells, indicative of a therapeutic window for re-activation of PTEN in BRAFi resistant melanoma cells. In support of this, we show that manipulation of the PTENP1-AS pathway re-sensitized the A375PR1 and A375VR4 resistant cells to treatment with vemurafenib (Fig. 5a,b and Supplementary Fig. S7).
We evaluated the expression of PTENP1-AS in regional lymph node metastases of stage III melanoma and found that high expression correlated with poor survival (Fig. 5d,e). This indicates that PTENP1-AS may be involved in melanoma tumor progression and is not only important during the development of resistance to BRAFi. Also, development of a particular type of resistance to vemurafenib may depend on the pre-existing factors/pathways in the tumors. High expression of PTENP1-AS could, for example, indicate that the tumor is less likely to respond to treatment and loss of PTEN expression has previously been linked to metastasis 13 . Therefore, one may speculate that PTENP1-AS is involved in such inactivation and consequently enhance initiation of distant metastasis. Additional studies will be required to better understand the functional role and importance of PTENP1-AS in melanoma progression and drug resistance i.e. by taking patient samples pre-and post-treatment with BRAFi.
In summary, we have identified PTENP1-AS to be involved both in melanoma tumor progression and in development of resistance to vemurafenib. The data presented here show that PTENP1-AS is not only a promising target for re-activation of PTEN, but also a possible prognostic marker for clinical outcome in stage III melanoma.
ChIP of EZH2, H3K27me3 and C/EBPB. ChIP assays were performed as previously described 8  www.nature.com/scientificreports/ 1:10 in ChIP dilution buffer, pre-cleared and incubated overnight with the appropriate antibody. Salmon sperm DNA/Protein A-agarose (Upstate/Millipore) was used to pull down the antibody. The DNA was eluted in elution buffer (1% SDS, 100 mM NaHCO 3 ), followed by reverse cross-linking at 65 °C overnight. The samples were RNase-A and protease-K treated and finally eluted using the Qiagen PCR purification kit (Qiagen Colony formation assay. 1000 cells per well were plated in 6 well plates and transfected 24 h later by using lipofectamine RNAiMax (Life Technologies), at a final concentration of 20 nM of gapmer ASO (Control or PTENP1-AS). Vemurafib treatment (or DMSO control) was initiated the day after transfection, cells were let grown for another 8-10 days before being fixed in 4% buffered formaldehyde and stained with 0.05% crystal violet. To estimate number of colonies, crystal violet staining was dissolved in 100% methanol, diluted to 1:10 in PBS and absorbance was measured at 540 nM using a Tecan Spark 10 M plate reader.

TCGA analysis. Gene expression profiles from GDC-TCGA (Genomic Data Commons-Cancer Genome
Atlas) were utilized to correlate the expression of PTENP1-AS and PTEN in skin cutaneous melanoma (SKCM). We used the Xena browser (https:// xenab rowser. net) to extract the RNA expression data for each gene, excluded samples with no expression of PTENP1-AS and PTEN, and correlated their expression using Spearman correlation (n = 251).
Patient samples. First regional lymph node metastases from stage III melanoma patients that had not received any systemic treatment were collected. The RNA was extracted using the Qiagen RNeasy mini kit (Qiagen). The RNA was DNase treated on column according to the manufacturer's recommendations. The expression of PTENP1-AS was analyzed using qRTPCR and standardized to Beta-actin (dCt) by using the corresponding oligos in Supplementary Table 1. ddCt was calculated by using the mean Ct value of all samples. High expression of PTENP1-AS (Fig. 5e) was defined as Ct < 30.

Statistics.
Two tailed Student's T-test was used to determine statistical significance. Error bars represent the standard error of the mean. Statistical significance of the melanoma patient samples in Fig. 5e is evaluated using log-rank test. Throughout the paper, *P < 0.05; **P < 0.01; ***P < 0.005.

Ethical approval and consent to participate. This study has been approved by the Research Ethics
Committee of Karolinska Institutet (Dnr: 2006/1373-31/3) and the Regional Ethics Committee in Lund (Dnr 2013/101). All involved individuals have provided informed consent to be included in the study. All the study procedures were carried out in accordance with relevant guidelines.