Aberrant expression of genes associated with stemness and cancer in endometria and endometrioma in a subset of women with endometriosis

STUDY QUESTION Is there molecular evidence for a link between endometriosis and endometriosis-associated ovarian cancers (EAOC)? STUDY ANSWER We identified aberrant gene expression signatures associated with malignant transformation in a small subgroup of women with ovarian endometriosis. WHAT IS KNOWN ALREADY Epidemiological studies have shown an increased risk of EAOC in women with ovarian endometriosis. However, the cellular and molecular changes leading to EAOC are largely unexplored. STUDY DESIGN, SIZE, DURATION CD73+CD90+CD105+ multipotent stem cells/progenitors (SC cohort) were isolated from endometrium (n = 18) and endometrioma (n = 11) of endometriosis patients as well as from the endometrium of healthy women (n = 14). Extensive phenotypic and functional analyses were performed in vitro on expanded multipotent stem cells/progenitors to confirm their altered characteristics. Aberrant gene signatures were also validated in paired-endometrium and -endometrioma tissue samples from another cohort (Tissue cohort, n = 19) of endometriosis patients. PARTICIPANTS/MATERIALS, SETTINGS, METHODS Paired-endometrial and -endometriotic biopsies were obtained from women with endometriosis (ASRM stage III–IV) undergoing laparoscopic surgery. Control endometria were obtained from healthy volunteers. Isolated CD73+CD90+CD105+ SC were evaluated for the presence of known endometrial surface markers, colony forming efficiency, multi-lineage differentiation, cell cycle distribution and 3D-spheroid formation capacity. Targeted RT-PCR arrays, along with hierarchical and multivariate clustering tools, were used to determine both intergroup and intragroup gene expression variability for stem cell and cancer-associated markers, in both SC+ and tissue cohorts. MAIN RESULTS AND THE ROLE OF CHANCE Isolated and expanded SC+ from both control and patient groups showed significantly higher surface expression of W5C5+, clonal expansion and 3D-spheroid formation capacity (P < 0.05) compared with SC−. The SC+ cells also undergo mesenchymal lineage differentiation, unlike SC−. Gene expression from paired-endometriosis samples showed significant downregulation of PTEN, ARID1A and TNFα (P < 0.05) in endometrioma compared with paired-endometrium SC+ samples. Hierarchical and multivariate clustering from both SC+ and tissue cohorts together identified 4 out of 30 endometrioma samples with aberrant expression of stem cell and cancer-associated genes, such as KIT, HIF2α and E-cadherin, altered expression ratio of ER-β/ER-α and downregulation of tumour suppressor genes (PTEN and ARID1A). Thus, we speculate that above changes may be potentially relevant to the development of EAOC. LARGE-SCALE DATA N/A. LIMITATIONS, REASON FOR CAUTION As the reported frequency of EAOC is very low, we did not have access to those samples in our study. Moreover, by adopting a targeted gene array approach, we might have missed several other potentially-relevant genes associated with EAOC pathogenesis. The above panel of markers should be further validated in archived tissue samples from women with endometriosis who later in life developed EAOC. WIDER IMPLICATIONS OF THE FINDINGS Knowledge gained from this study, with further confirmation on EAOC cases, may help in developing screening methods to identify women with increased risk of EAOC. STUDY FUNDING/COMPETING INTEREST(S) The study is funded by the Swedish Research Council (2012-2844), a joint grant from Stockholm County and Karolinska Institutet (ALF), RGD network at Karolinska Institutet, Karolinska Institutet for doctoral education (KID), Estonian Ministry of Education and Research (IUT34-16), Enterprise Estonia (EU48695), Horizon 2020 innovation program (WIDENLIFE, 692065), European Union’s FP7 Marie Curie Industry-Academia Partnerships and Pathways funding (IAPP, SARM, EU324509) and MSCA-RISE-2015 project MOMENDO (691058). All authors have no competing interest

<i>GSTM1</i> Gene Expression Correlates to Leiomyoma Volume Regression in Response to Mifepristone Treatment

<div><p></p><p>Progesterone receptor modulators, such as mifepristone are useful and well tolerated in reducing leiomyoma volume although with large individual variation. The objective of this study was to investigate the molecular basis for the observed leiomyoma volume reduction, in response to mifepristone treatment and explore a possible molecular marker for the selective usage of mifepristone in leiomyoma patients. Premenopausal women (N = 14) were treated with mifepristone 50 mg, every other day for 12 weeks prior to surgery. Women were arbitrarily sub-grouped as good (N = 4), poor (N = 4) responders to treatment or intermediate respondents (N = 3). Total RNA was extracted from leiomyoma tissue, after surgical removal of the tumour and the differential expression of genes were analysed by microarray. The results were analysed using Ingenuity Pathway Analysis software. The glutathione pathway was the most significantly altered canonical pathway in which the glutathione-s transferase mu 1 (<i>GSTM1</i>) gene was significantly over expressed (+8.03 folds) among the good responders compared to non responders. This was further confirmed by Real time PCR (p = 0.024). Correlation of immunoreactive scores (IRS) for GSTM1 accumulation in leiomyoma tissue was seen with base line volume change of leiomyoma R = −0.8 (p = 0.011). Furthermore the accumulation of protein GSTM1 analysed by Western Blot correlated significantly with the percentual leiomyoma volume change R = −0.82 (p = 0.004). Deletion of the <i>GSTM1</i> gene in leiomyoma biopsies was found in 50% of the mifepristone treated cases, with lower presence of the GSTM1 protein. The findings support a significant role for GSTM1 in leiomyoma volume reduction induced by mifepristone and explain the observed individual variation in this response. Furthermore the finding could be useful to further explore GSTM1 as a biomarker for tailoring medical treatment of uterine leiomyomas for optimizing the response to treatment.</p><p>Clinical Trials identifier</p><p><a href="http://www.clinicaltrials.gov" target="_blank">www.clinicaltrials.gov</a>: NCT00579475, Protocol date: November 2004. <a href="http://clinicaltrials.gov/ct2/show/NCT00579475" target="_blank">http://clinicaltrials.gov/ct2/show/NCT00579475</a></p></div

Correlation of leiomyoma volume change (percentage ×0.1) and the expression levels of GSTM1 as studied by real time PCR and Western blot GR = good responders, PR = poor responders, ND = not determined (in between groups) category.

<p>Correlation of leiomyoma volume change (percentage ×0.1) and the expression levels of GSTM1 as studied by real time PCR and Western blot GR = good responders, PR = poor responders, ND = not determined (in between groups) category.</p

Differentially expressed genes, with mifepristone treatment: Genes, their cellular location and the fold changes with mifepristone treatment on leiomyoma patients as analysed by microarray followed by IPA.

<p>Differentially expressed genes, with mifepristone treatment: Genes, their cellular location and the fold changes with mifepristone treatment on leiomyoma patients as analysed by microarray followed by IPA.</p

Flow chart explaining the patient enrolment, allotment and relevent details, as per consolidated standards of reporting trials (CONSORT) in the study where leiomyoma patients were treated with mifepristone/placebo.

<p>Flow chart explaining the patient enrolment, allotment and relevent details, as per consolidated standards of reporting trials (CONSORT) in the study where leiomyoma patients were treated with mifepristone/placebo.</p

GSTM 1 expression leiomyoma volume reduction: Comparison of GSTM1 expression as seen by by real time PCR and Western Blot in relation to percentage of leiomyoma volume change during 3 months of mifepristone treatment.

<p>GR-good responders (>30% volume reduction); PR-poor responders (<18% volume reduction).</p>*<p>no gene expression detected.</p

Glutathione pathway gene expression and leiomyoma volume reduction: Expression levels and fold changes of differentially expressed genes in glutathione pathway in good and poor responders for leiomyoma patients with 12 weeks of mifepristone treatment as studied by with 12 weeks of mifepristone treatment in leiomyoma tissue as studied by real time PCR and microarray (*The least responder did not have any detectable Ct value for GSTM1).

<p>Glutathione pathway gene expression and leiomyoma volume reduction: Expression levels and fold changes of differentially expressed genes in glutathione pathway in good and poor responders for leiomyoma patients with 12 weeks of mifepristone treatment as studied by with 12 weeks of mifepristone treatment in leiomyoma tissue as studied by real time PCR and microarray (*The least responder did not have any detectable Ct value for GSTM1).</p

Accumulation of GSTM1 protein in GR (good responders) or PR (poor responders to mifepristone treatment as demonstrated by Western Blot.

<p>Accumulation of GSTM1 protein in GR (good responders) or PR (poor responders to mifepristone treatment as demonstrated by Western Blot.</p