HOTAIR and its surrogate DNA methylation signature indicate carboplatin resistance in ovarian cancer

Background Understanding carboplatin resistance in ovarian cancer is critical for the improvement of patients’ lives. Multipotent mesenchymal stem cells or an aggravated epithelial to mesenchymal transition phenotype of a cancer are integrally involved in pathways conferring chemo-resistance. Long non-coding RNA HOTAIR (HOX transcript antisense intergenic RNA) is involved in mesenchymal stem cell fate and cancer biology. Methods We analyzed HOTAIR expression and associated surrogate DNA methylation (DNAme) in 134 primary ovarian cancer cases (63 received carboplatin, 55 received cisplatin and 16 no chemotherapy). We validated our findings by HOTAIR expression and DNAme analysis in a multicentre setting of five additional sets, encompassing 946 ovarian cancers. Chemo-sensitivity has been assessed in cell culture experiments. Results HOTAIR expression was significantly associated with poor survival in carboplatin-treated patients with adjusted hazard ratios for death of 3.64 (95 % confidence interval [CI] 1.78–7.42; P < 0.001) in the discovery and 1.63 (95 % CI 1.04–2.56; P = 0.032) in the validation set. This effect was not seen in patients who did not receive carboplatin (0.97 [95 % CI 0.52–1.80; P = 0.932]). HOTAIR expression or its surrogate DNAme signature predicted poor outcome in all additional sets of carboplatin-treated ovarian cancer patients while HOTAIR expressors responded preferentially to cisplatin (multivariate interaction P = 0.008). Conclusions Non-coding RNA HOTAIR or its more stable DNAme surrogate may indicate the presence of a subset of cells which confer resistance to carboplatin and can serve as (1) a marker to personalise treatment and (2) a novel target to overcome carboplatin resistance. Electronic supplementary material The online version of this article (doi:10.1186/s13073-015-0233-4) contains supplementary material, which is available to authorized users.


Background
Late stage presentation aside, carboplatin resistance in ovarian cancer is the key obstacle to improving survival in this disease [1]. The observation that re-treatment with platinum-based drugs 6-12 months after primary response proved to be successful in a certain percentage of patients [2] is consistent with the idea that platinum sensitivity can be modulated by both cancer cell-autonomous and non-autonomous factors. For both these factors stromal/mesenchymal differentiation is crucially important. Epithelial-mesenchymal transition (EMT) in ovarian cancer cells is associated with platinum resistance [3][4][5]. On the other hand the cancer cell-autonomous tumour stroma and mesenchymal stem cells (MSCs)mainly recruited from the bone marrow [6] might play an important role in ovarian cancer biology [7,8]. Recently, bone marrow-derived MSCs and embryonic fibroblasts, but not more extensively differentiated stromal cells, have been shown to induce platinum resistance in ovarian cancer [9].
Long non-coding RNAs are known to epigenetically remodel chromatin states and influence gene transcription in normal and cancer tissue towards stromal/mesenchymal differentiation [10][11][12][13]. Aberrant expression of non-coding RNAs has been observed in numerous diseases, including cancer [14], yet their precise contribution to disease aetiology and biology is far from clear. HOX antisense transcript intergenic RNA (HOTAIR), transcribed from the HOXC locus, represses transcription by recruiting polycomb repressive complex 2 (PRC2) to specific polycomb group target (PCGT) genes, in particular to those normally targeted by PRC2 in embryonic fibroblasts [10].
In stem cells, PCGTs are repressed through PRC2 occupancy and PCGTs important for specialised cell identities become de-repressed upon differentiation [15,16]. We and others have shown that the promoters of these stem cell PCGTs become methylated and silenced in cancer [17][18][19][20]. It was recently reported that the expression of HOTAIR is increased in various cancer entities and that high levels of expression correlate with cancer invasiveness, metastases and poor prognosis [10,21]. A recent systematic review of 19 papers (including a total of 2255 patients) demonstrates consistently that HOTAIR expression is a poor prognostic marker across a large set of cancers [22]. It is unclear, however, whether HOTAIR is associated with an aberrant DNA methylation profile in cancer and whether this robust DNA-based imprint mediates resistance to specific drugs.
Here we tested the hypothesis that HOTAIR RNA expression, or a HOTAIR-associated DNA methylation (DNAme) signature, as surrogates for mesenchymal differentiation, serve as markers for carboplatin resistance in primary ovarian cancer.

Ovarian cancer data sets
We analyzed six different data sets, details of which are provided in Additional file 1.
The first data set consisted of primary ovarian cancer samples (n = 134, 24-87 years, median 62.7 years at diagnosis; Additional file 2) treated at the Innsbruck Medical University, denoted "INNSBRUCK". Clinicopathological features are shown in Table 1. The study was approved by the ethical committee of the Medical University Innsbruck (reference number UN4044). For the majority of patients exemption from obtaining informed consent was received as the majority of ovarian cancer patients were dead at the time the application was evaluated. The median survival time was 3.8 years.
HOTAIR expression was measured in all 134 samples. DNA methylation data are available as Additional file 3.
The second data set consisted of primary ovarian cancer samples (n = 175, 21-83 years, median 60.0 years at diagnosis; Additional file 4) treated at the University Medical Center in Groningen [23], denoted "GRO-NINGEN". The median survival time in this set, which consisted only of stage III/IV patients, was 2.1 years. For these 175 samples, we measured HOTAIR expression. Of the 175 samples, 157 received carboplatin only, whilst 18 received cisplatin instead. For 114 of these samples . Patients gave informed consent for collection and storage of tissue samples in a tissue bank for future research. All relevant patient data were retrieved and transferred into an anonymous, passwordprotected database. The patients' identity was protected by study-specific, unique patient codes and their true identity was only known to two dedicated data managers. According to Dutch regulations, these precautions meant no further institutional review board approval was needed.
The third data set consisted of primary ovarian cancer samples (n = 342, serous ovarian cancers, median 58 years at diagnosis; 316 received carboplatin-based chemotherapy with the rest received cisplatin or were untreated), analyzed within The Cancer Genome Atlas (TCGA) program, and for which Illumina Infinium 27 k DNAme data were publicly available [24] (Additional file 5), denoted "TCGA". The median survival time was 2.6 years.
The fourth data set consisted of primary ovarian cancer samples from three European Cancer centres (Leuven, Oslo, Rotterdam; n = 206, median 58 years at diagnosis; Additional file 6), denoted "EUROPE". The median survival time was 3.4 years. For this data set, Illumina Infinium 450 k DNAme data were available for 121 carboplatin-and 85 cisplatin-treated patients. Data are deposited in the GEO, accession [GEO:GSE72021]. The study from Rotterdam has been approved by the local medical ethics committee (MEC-2008-183), performed in accordance with the Code of Conduct of the Federation of Medical Scientific Societies in the Netherlands. The Regional Committee for Medical Research Ethics in Norway approved the study (for patients diagnosed before 2007, exemption from obtaining informed consent was received as the majority of ovarian cancer patients were dead at the time the application was evaluated; patients diagnosed after 2007 signed general consent allowing for use of the tumours for research purposes). Written informed consent for the use of tumour tissue and prospective clinical data collection was obtained from all patients and approved by the Leuven ethics committee.
The fifth data set consisted of primary ovarian cancer samples from Bergen (n = 49) with 40 receiving carboplatin and 9 untreated (no chemotherapy), denoted "BERGEN". Patients were included in the study after written informed consent, approved by the Regional Research Ethics Committee in Medicine. For samples from this cohort, we measured HOTAIR expression.
The sixth data set consisted of primary ovarian cancer samples from Rochester-Mayo (n = 174), denoted "ROCHESTER-MAYO". All 174 patients received carboplatin and for these samples we measured DNAme using Illumina 450 k beadarrays. All patients gave informed consent and the Mayo Clinic Institutional Review Board approved the study. The data are available from Dr Ellen Goode at the Department of Health Sciences Research, Mayo Clinic, Rochester, USA, upon request.
Our research conformed to the Helsinki Declaration.

HOTAIR expression
Total RNA was extracted by the acid guanidium thiocyanate-phenol-chloroform method [25]. Reverse transcription of RNA was performed as previously described [26]. Primers and probes for HOTAIR were designed using Primer Express (Applied Biosystems, Foster City, CA, USA). Samples in which HOTAIR was not amplified by real-time PCR after 45 cycles were classified as negative (HOTAIR-ve; Additional file 1).

HOTAIR overexpression in ovarian cancer cell lines
The SKOV3IP cells were stably transduced with HOTAIR and LacZ constructs, kindly provided by Dr Chang (Stanford) [10] and single clones of HOTAIR/ LacZ overexpressing cells were used for experiments (Additional file 1). Cells were treated with cisplatin (0.5-18 μM) or carboplatin (10-160 μM) for 3 days and analysed by the cell survival MTT assay (Sigma).

Statistics
To test for differences in categorical variables, we used the Chi square test. Impact of HOTAIR expression on ovarian cancer survival was ascertained using log rank test and Kaplan Meier curves. To demonstrate the effect of HOTAIR expression on DNAme patterns, we first performed univariate analysis to rank CpGs according to their association with HOTAIR expression (Additional file 1). We used 10-fold internal cross-validations to identify an optimal HOTAIR-associated DNAme signature consisting of 67 CpGs (Additional file 7) at an estimated false discovery rate (FDR) of approximately 0.17.
To build a single-sample classifier from this signature, a cutoff was optimized using receiver operating characteristic (ROC) analysis. This same cutoff was then used to assign samples from independent cohorts into two groups exhibiting high and low correlations with the HOTAIR DNAme signature. For the PCGT enrichment analysis we relaxed the threshold of the DNAme signature to include the top 500 ranked CpGs (FDR < 0.3), and divided the 500 into the 233 which were hypermethylated and the 267 which were hypomethylated in high HOTAIR expressors.

Results
HOTAIR expression in primary ovarian cancer is not associated with clinicopathological features We analyzed HOTAIR expression in 134 primary ovarian cancer samples (INNSBRUCK) and found that 72 were positive and 62 were negative for HOTAIR.  Only 34 % of patients who received carboplatin and whose tumours expressed HOTAIR survived the first 3 years post-diagnosis, whereas 85 % of those patients who received carboplatin, but whose tumours were HOTAIR-ve, survived during the same time period (Fig. 1a). Nearly half of the carboplatin-treated patients also received paclitaxel, and in both groups, i.e., single agent carboplatin and combined carboplatin/paclitaxel, HOTAIR was significantly associated with poor outcome (log rank P value = 0.006 and 0.003, respectively), again indicating the interaction of HOTAIR with carboplatin but not with cisplatin or paclitaxel.
To validate these findings we analyzed 175 ovarian cancer samples from Groningen [23] and 49 samples from Bergen and again confirmed that HOTAIR expression is a poor prognostic factor specifically in carboplatin-treated patients (Fig. 1c, d; Additional files 9 and 10).

HOTAIR expression is associated with a DNAme profile enriched for PCGTs and associated with multipotent MSCs
As HOTAIR is known to modulate chromatin, in particular at PCGTs, we asked if DNAme differed between HOTAIR-expressing and non-expressing ovarian cancer samples. Since HOTAIR expression was associated with carboplatin resistance, we restricted the analysis to the 63 carboplatin-treated patients in the INNSNRUCK set. Of these 63 samples, 35 expressed HOTAIR whilst 28 did not. We identified a 67-CpG DNAme signature representing a statistically significant association with HOTAIR expression. Boxplots of beta methylation values of the 67 CpGs confirmed the relatively large differences in methylation between HOTAIR expressors and non-expressors (Additional file 11). We observed that many of the top CpGs mapped to PCGTs (Fig. 2a) and found that PCGTs (defined as PRC2 targets in both human embryonic stem cells and human embryonic fibroblasts, but not PRC2 targets in breast cancer cells) were highly enriched among CpGs hypermethylated in HOTAIR expressors (Additional file 12). HOTAIR expression was also associated with lower expression of PCGTs, in particular human embryonic fibroblast PCGTs, in the GRONINGEN set [23], supporting the role of HOTAIR as an epigenetic regulator of MSCs in ovarian cancer (Additional file 13). By correlating the 67-CpG DNAme signature to the methylation profile of any given sample, a correlation score was obtained which can be viewed as a DNA-based surrogate for HOTAIR expression. We first evaluated this correlation score in early passage (multipotent) MSCs, late passage (more differentiated) MSCs, reprogrammed MSCs, embryonic stem cells [29] and ovarian cancer cell lines, demonstrating that the HOTAIR DNAme signature is likely to be a surrogate marker for either multipotent MSCs within the ovarian cancer tissue, or for ovarian cancer cells with an increased tendency to undergo EMT ( Fig. 2b; Additional file 14).
The HOTAIR-DNAme signature predicts survival outcome Given the relationship between HOTAIR expression and carboplatin resistance, we next checked that the HOTAIR DNAme signature would be similarly associated with outcome in the 63 carboplatin-treated subgroup in the INNSBRUCK set. As expected, the score obtained by correlating the sample-specific DNAme profile to the HOTAIR DNAme signature was predictive of carboplatin resistance in univariate as well as in multivariate Cox-regression analyses adjusted for age, stage and size of residual tumour (Additional file 15). In order to build a single-sample classifier we also optimized a cutoff on the correlation score of the ovarian cancer tissue samples using ROC analysis to ensure approximately 80 % sensitivity and 80 % specificity between the DNAme signature and HOTAIR expression (Additional file 16). Dividing the samples into two groups based on this cutoff further confirmed a significant difference in survival rates of these two subgroups of patients, i.e., samples with a high DNAme signature score had a HR of 3.07 (P = 0.001) for death relative to those with a low surrogate score (Fig. 2c).

Validation of HOTAIR DNAme signature in three independent large cohorts
To validate our HOTAIR DNAme signature, we first tested it in an independent large data set of serous ovarian cancers from TCGA set, consisting of 316 patients who received carboplatin-based therapy and 26 who received cisplatin. For each of the carboplatintreated patients, we computed a carboplatin resistance score by correlating the DNAme profile of the tumour to the previously determined 67-CpG DNAme HOTAIR signature. This score predicted outcome in both univariate as well as multivariate Cox-regression analyses (Additional file 15), and was a much stronger predictor of outcome than scores constructed using random signatures (Additional file 17). Using the previously determined cutoff to assign samples into high and low DNAme signature score groups further demonstrated the robustness of the predictive classifier in the carboplatin-treated subgroup (Fig. 2d). Of note, the prediction obtained using the HOTAIR DNAme signature outperforms the classification obtained using either mRNA or microRNA expression predictors, as reported in TCGA study [24]. In contrast to the carboplatin-treated group, the HOTAIR DNAme signature was not predictive of outcome in the 26 patients in TCGA set who did not receive carboplatin (Fig. 2e). We further tested the HOTAIR DNAme signature in an independent set of 121 carboplatin-treated patients (EUROPE set) with DNAme profiles generated using a different assay (Illumina Infinium Human Methylation 450 k). In order to more rigorously assess the specificity of the signature, it was also tested in a further 85 cisplatin-treated patients from the same EUROPE cohort which were also profiled with the same 450 k technology. Once again, the DNAme signature-based surrogate scores for HOTAIR expression correctly predicted carboplatin resistance (Fig. 2f; Additional file 18), with no association observed in the cisplatin-treated subgroup ( Fig. 2g; Additional file 18).
Further strengthening the robustness of the HOTAIR DNAme signature, we found it to be predictive of carboplatin resistance in an additional independent set of 174 carboplatin-treated ovarian cancer patients (ROCHES-TER-MAYO set), which had also been profiled with Illumina 450 k DNAme bead arrays (Additional file 19).

HOTAIR expressors respond preferentially to cisplatinbased chemotherapy
In all data sets analysed, we observed a consistent trend for HOTAIR expression, or DNAme-based surrogate HOTAIR expression, to be associated preferentially with cisplatin response, although statistical significance was not observed in individual data sets. Thus, to investigate this further we used a meta-analysis approach and asked if the type of chemotherapy received was associated with a different response in HOTAIR expressors compared with non-expressors (Table 2). In all data sets the risk of death was lower in HOTAIR expressors who received cisplatin compared with those who received carboplatinbased therapies, whereas for non-HOTAIR expressors the opposite pattern was observed (Table 2; Additional files 20 and 21). Using a combined probability Fisher test in a meta-analysis over all data sets, we found a highly significant interaction between chemotherapy type received and HOTAIR expression in dictating response to treatment (P < 0.001; Table 2), which was retained in multivariate analysis (P = 0.008; Table 2).

Effect of HOTAIR expression and platinum sensitivity in ovarian cancer cell line
In order to test whether HOTAIR expression modulates response to carboplatin we used SKOV3IP ovarian cancer cells, which are sensitive to platinum-based chemotherapy and do not express HOTAIR. We observed that overexpression of HOTAIR in this particular cell line reduces only sensitivity to carboplatin but not cisplatin (Fig. 3). The half maximal inhibitory concentration (IC 50 ) shifted from 30 to 60 μM for carboplatin and from 3 to 3.5 μM for cisplatin in LacZ and HOTAIR-expressing SKOV3IP cells, respectively. HOTAIR expression on two other cell lines had either no effect (A2780) or increased cisplatin sensitivity (OVCAR8) (Additional file 22).

Discussion
Here we have shown that women with ovarian tumours expressing HOTAIR RNA, or an equivalent HOTAIR-associated DNAme signature, experience a poorer survival outcome post carboplatin-based chemotherapy compared with HOTAIR-ve tumours.
These data offer solid evidence for two novel concepts. First, we demonstrate that not only a non-coding RNA, but importantly also a presumed downstream effect, represented by a specific DNAme signature, reproducibly predicts cancer outcome. This concept is appealing, because (i) a DNA-based biomarker is more stable and (ii) would provide a spatially and temporally more comprehensive surrogate for cancer biology compared with a snap-shot RNA assessment. Second, our data very much challenge the dogma that cisplatin and carboplatin have the same effect on ovarian cancer and that parameters that allow for discrimination between patients that benefit form carboplatin and those that benefit form cisplatin would not exist.
A recent study from Roodhart et al. [9] highlights the complexity of the chemo-resistance response which (See figure on previous page.) Fig. 2 HOTAIR-associated DNA methylation signature predicts survival in carboplatin-treated ovarian cancer patients. a Heatmap of the 67 CpG DNAme signature (gene symbol for PCGTs in red) strongly associated with HOTAIR expression in ovarian cancers. CpG methylation profiles were centred to mean zero and scaled to unit variance. Blue denotes relative high methylation, yellow relative low methylation. b Correlation scores of the 67-CpG HOTAIR DNAme signature with the corresponding DNAme profiles in embryonic stem cells (ESC), reprogrammed MSCs (iPSC; induced pluripotent stem cells), ovarian cancer cell lines (OVC) with and without stable expression of HOTAIR and MSCs harvested at late (more than four passages) or at early passage (fewer than four passages); P = 10 −6 comparing all MSCs to combined ESC/iPSC, P = 10 −5 comparing all OVC to all MSC. c Kaplan-Meier curves and log rank test P value of carboplatin-treated patients with a high and low DNAme HOTAIR signature score (INNSBRUCK set). d Validation of the DNA methylation HOTAIR signature in an independent large set of carboplatin-treated serous ovarian cancers from TCGA set (n = 316). e Corresponding Kaplan-Meier curve of HOTAIR signature predictions in the non-carboplatin-treated subset of the TCGA set (n = 26). f Validation of the DNA methylation HOTAIR signature in an independent large set of carboplatin-treated ovarian cancers (EUROPE set, n = 121). g Corresponding Kaplan-Meier curve of HOTAIR signature predictions in the non-carboplatin (cisplatin) treated subset (EUROPE set, n = 85), validating the specificity of the signature. HA HOTAIR, OS overall survival integrally involves cancer cell non-autonomous factors. The authors used a mouse model and demonstrated that cells with a multilineage differentiation potential, such as embryonic fibroblasts, become activated during treatment with platinum analogs and secrete factors systemically that protect tumour cells against platinum chemotherapeutics. Roodhart et al. [9] demonstrated this for both carboplatin and cisplatin whereas our databased on 1080 human ovarian cancer samplesdemonstrate that HOTAIR and its surrogate DNAme signature are associated only with carboplatin resistance and not cisplatin resistance.
Although in the past cisplatin and carboplatin have been considered to be nearly identical drugs and prospective randomized trials showed similar overall survival for cisplatin-and carboplatin-based regimens in ovarian cancer [30,31], both drugs have substantially different side-effect profiles with higher rates of nausea, vomiting and renal toxicity for cisplatin and thrombocytopenia for carboplatin [32]. Moreover, cisplatin and carboplatin are known to differ in their cell membrane transport characteristics [33]. This is noteworthy because cell membrane transport proteins have been shown to be critical determinants of platinum drug sensitivity/resistance, possibly as a result of secreted factors from MSCs which can affect transport characteristics. Lending further credence to our data, in cervical cancer (a disease which is known to expresses high levels of HOTAIR [34]) carboplatin has been repeatedly reported to be a less effective platinum analog than cisplatin [35][36][37].  Although we provide unprecedented strong evidence for a non-coding RNA and its DNAme surrogate signature to be a predictive and prognostic marker in ovarian cancer, there are several limitations to our study. First, although in situ hybridisation for HOTAIR has been successfully established for cell lines, we were not able (despite substantial efforts; data not shown) to determine the specific subset of HOTAIR-expressing cells within the bulk tumour sample. Hence, at this stage we can only state that HOTAIR expression serves as an excellent surrogate for "mesenchyme-ness" of a cancer, not knowing whether this reflects the presence of MSCs in the tumour stroma or whether it is a reflection of the number of cancer cells that have undergone EMT, or a combination of both. Second, we don't provide a mechanistic model as to why and how HOTAIR modulates carboplatin and cisplatin response differently. Whereas recent evidence shows that HOTAIR promotes proliferation by modulating cell cycle and apoptosis [38], no evidence exists to show that this has an impact on platinum resistance. Our findings are consistent with the view that HOTAIR modulates the epigenome at the level of the DNA methylome in both cancer cells and tumour stroma. Although still speculative, it is likely that the differential response of carboplatin and cisplatin is due to underlying differences in how the MSC biology of tumour stroma and the EMT characteristics of cancer cells affect the two drugs. Support for this view comes from recent data (unpublished): we have analysed MSCs and modulated expression of HOTAIR. Overexpression and knockdown of HOTAIR inhibited or stimulated, respectively, in vitro differentiation of MSCs. Modification of HOTAIR expression evoked consistent effects on gene expression, particularly in polycomb group target genes and genes involved in cancer. Furthermore, overexpression and knockdown of HOTAIR resulted in DNAme changes that are enriched in HOTAIR binding sites.
Despite these limitations, our findings have a number of immediate clinical implications. We provide a solid rationale for prospective randomized clinical trialsideally in a neo-adjuvant settingto assess whether the HOTAIR DNAme signature is an appropriate tool to stratify women with ovarian cancer (and possibly also other cancers) into groups which benefit preferentially from cisplatin or from carboplatin treatment. Strategies to reduce HOTAIR activity (i.e., by intra-peritoneal HOTAIR small interfering RNA) may lead to a novel strategy to (re)sensitize cancers to chemotherapy.