A novel role of the Sp/KLF transcription factor KLF11 in arresting progression of endometriosis.

Endometriosis affects approximately 10% of young, reproductive-aged women. Disease associated pelvic pain; infertility and sexual dysfunction have a significant adverse clinical, social and financial impact. As precise disease etiology has remained elusive, current therapeutic strategies are empiric, unfocused and often unsatisfactory. Lack of a suitable genetic model has impaired further translational research in the field. In this study, we evaluated the role of the Sp/KLF transcription factor KLF11/Klf11 in the pathogenesis of endometriosis. KLF11, a human disease-associated gene is etiologically implicated in diabetes, uterine fibroids and cancer. We found that KLF11 expression was diminished in human endometriosis implants and further investigated its pathogenic role in Klf11-/- knockout mice with surgically induced endometriotic lesions. Lesions in Klf11-/- animals were large and associated with prolific fibrotic adhesions resembling advanced human disease in contrast to wildtype controls. To determine phenotype-specificity, endometriosis was also generated in Klf9-/- animals. Unlike in Klf11-/- mice, lesions in Klf9-/- animals were neither large, nor associated with a significant fibrotic response. KLF11 also bound to specific elements located in the promoter regions of key fibrosis-related genes from the Collagen, MMP and TGF-β families in endometrial stromal cells. KLF11 binding resulted in transcriptional repression of these genes. In summary, we identify a novel pathogenic role for KLF11 in preventing de novo disease-associated fibrosis in endometriosis. Our model validates in vivo the phenotypic consequences of dysregulated Klf11 signaling. Additionally, it provides a robust means not only for further detailed mechanistic investigation but also the ability to test any emergent translational ramifications thereof, so as to expand the scope and capability for treatment of endometriosis

Role of Klf11 on lesion size in endometriosis.

<p>(<b>A</b>) Endometriosis was surgically induced in 8 week old <i>Klf11-/-</i> and wildtype female mice (N = 7/group). All animals were weighed prior to initial surgery and at necropsy 3 weeks later. There was no difference in weight between the groups either prior to implantation surgery or before subsequent necropsy. Comparative weight profile prior to necropsy shown (p>0.05; NS). (<b>B, C</b>) At induction, 0.5cm endometrial implants were implanted on to the parietal peritoneum of <i>Klf11-/-</i> and wildtype mice. Lesion size was evaluated at necropsy three weeks after initial surgery. The peritoneal lesions (white arrows; box) in <i>Klf11-/-</i> mice (C) were larger and more cystic compared to those observed in wildtype controls (B). (<b>D</b>) The lesions in <i>Klf11-/-</i> animals (6.8±0.044mm) were significantly larger than those observed in wildtype controls (4.5 ± 0.029mm). [*  = p<0.05; 14 lesions per genotype].</p

Comparison of the role of Klf9 and Klf11 in an animal endometriosis model.

<p>(<b>A</b>) Endometriotic Lesions (circled) in <i>Klf9-/-</i> animals were either unchanged or had regressed in size from the time of initial peritoneal implantation, when evaluated at necropsy 3 weeks later. (<b>B</b>) Endometriotic lesions in these animals also did not elicit a progressive fibrotic response as seen in <i>Klf11-/-</i> animals. Any adhesions in <i>Klf9-/-</i> animals (black arrow) were flimsy, transparent and peri-lesional in extent. (<b>C</b>) Tissue planes were unaltered with preservation of intra-abdominal anatomy. Consequently, intestinal length was not foreshortened due to lack of mesenteric fibrosis (unraveled intestinal loops denoted by white arrows). (<b>D</b>): A composite adhesion score for each mouse was determined and compared between <i>Klf11-/-</i>, <i>Klf9-/-</i> and wildtype genotypes, based on the Murine Adhesion Scoring System. The adhesion score for <i>Klf11-/-</i> mice (81.7±4.8) was significantly different from that calculated in either <i>Klf9-/-</i> (12.3±1.8) or wildtype animals (9.17±0.8); (* = p<0.05, 14 lesions/genotype). The scores objectively reflected observed anatomical findings in these animals.</p

Primer Sequences used for amplification of promoter sequences (ChIP) or expression studies (PCR).

<p>Primer Sequences used for amplification of promoter sequences (ChIP) or expression studies (PCR).</p

Application of a Novel Fibrosis Adhesion Scoring System to evaluate murine endometriotic lesions.

<p>A murine peritoneal sclerosis scoring system was modified and adapted to the revised ASRM endometriosis staging system. Accordingly, anatomic landmarks in the region of the lesions were incorporated and weighted scores were assigned in accordance with the established human disease scoring system.</p

KLF11 expression in human urogenital tissues, uterine eutopic endometrium and endometriosis.

<p>(<b>A</b>) Pooled Human Organ-specific RNA (1 µg/reaction) was analyzed for <i>KLF11</i> mRNA expression by PCR. mRNA levels of the housekeeping gene <i>GAPDH</i> were simultaneously assessed as loading control. Relative organ-specific <i>KLF11</i> mRNA expression levels were determined by densitometric comparison to corresponding <i>GAPDH</i> levels. KLF11 mRNA expression levels were increased in urogenital tissues such as the kidney, ovary, placenta, testes and uterus (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060165#pone-0060165-g001" target="_blank">Figure 1A</a>, arrows) compared to other non-urogenital tissues. (<b>B</b>) Expression of KLF11 mRNA and protein were also assessed in two cell lines Ishikawa, a well-differentiated endometrial adenocarcinoma cell line and Human Endometrial Stromal Cells (T-HESC), which are frequently used as model endometrial epithelial and stromal cell lines respectively. <i>KLF11</i>/KLF11 were expressed in both cell-lines; <i>GAPDH/</i>α-TUBULIN were used as a reference controls. To qualitatively demonstrate KLF11 expression in each of these endometrial cell lines, lysate for RNA or protein extraction was obtained from one 10-cm cell culture dish of confluent cells. Due to much lower abundance of stromal compared to epithelial cells there appears to be differential <i>KLF11</i>/KLF11 expression between the cell-types. However, this difference was also reflected in expression of the loading controls <i>GAPDH</i>/α-TUBULIN between the two cell lines. (<b>C, D</b>) KLF11 expression was also evaluated by immunohistochemistry in eutopic endometrium as well as in ectopic endometrial implants (magnification: 200×: panel; 400×: inset). KLF11 was expressed in the nuclei and cytoplasm of epithelial as well as stromal cells in both eutopic endometrium as well as in endometriotic implants. KLF11 expression was diminished in endometriotic implants compared to eutopic endometrium. Representative samples shown. (<b>E</b>) KLF11 expression levels were compared and scored in paired eutopic endometrium and ectopic endometriotic implants obtained from the same patient (N = 28 paired samples). The expression was significantly reduced in the implants (epithelium: 103±2.6; stroma: 40.4±3.9) compared to that in eutopic uterine endometrium (epithelium: 278±2.5; stroma: 114±8.7). * and. = p<0.05 and represent comparisons between epithelial and stromal cells in eutopic and ectopic endometrium respectively.</p

Role of Klf11 in endometriosis-associated fibrosis.

<p>Endometriotic lesions in <i>Klf11-/-</i> mice were associated with prolific <i>de novo</i> scar tissue formation in contrast to wildtype controls. (<b>A</b>) Adhesions in <i>Klf11-/-</i> animals were thick, opaque, dense and unyielding to mechanical disruption by pressure. The adhesions had a broad base (black arrows) and involved adjacent viscera such as the small and large intestine, stomach and liver, thereby resulting in obliteration of physiological tissue planes. (<b>B</b>) Progressive fibrosis further involved the intestinal mesentery in these animals, resulting in straightening of the bowel with apparent shortening of length (white arrows). (<b>C</b>) In contrast, in wildtype animals, the lesions remained discrete (white arrows and box in C and D) with minimal adhesions (black arrow). Any adhesions that formed were slender, transparent, non-obliterating and very easily disrupted by pressure. (<b>D</b>) Lack of progressive and prolific fibrosis in wildtype animals preserved normal intra-abdominal anatomy with no peritoneal obliteration or mesenteric fibrosis.</p

Evaluation of the Role of KLF11 in the regulation of fibrogenic signaling.

<p>(<b>A</b>) KLF11 binding to the promoters of known fibrosis associated genes was evaluated by Chromatin immunoprecipitation <i>in vivo</i> in endometrial stromal cells. Representative targets from diverse families of fibrogenic genes are shown (Collagens, MMP, TGF-β signaling pathway). KLF11 but not a control species and isotype-specific IgG bound candidate promoter GC-elements in these putative target genes. (<b>B</b>) Functional competence of the KLF11-binding promoter element was tested in promoter-luciferase reporter assays. T-HESC endometrial cells were transfected with 2.5 µg of either pcDNA3/His-<i>KLF11</i> or corresponding pcDNA3/His-<i>EV</i> and 3 µg of pGL3/promoter-reporter construct for 48 hours. Normalized luciferase expression levels obtained with KLF11 compared to EV are shown. Compared to corresponding empty vector, KLF11 significantly repressed COL1A1, 1A2, MMP3, 10 and TGFβR1- promoter luciferase levels, whereas it activated expression from the COL3A1-reporter (* = p<0.05).</p

LPCAT1-TERT fusions are uniquely recurrent in epithelioid trophoblastic tumors and positively regulate cell growth.

Gestational trophoblastic disease (GTD) is a heterogeneous group of lesions arising from placental tissue. Epithelioid trophoblastic tumor (ETT), derived from chorionic-type trophoblast, is the rarest form of GTD with only approximately 130 cases described in the literature. Due to its morphologic mimicry of epithelioid smooth muscle tumors and carcinoma, ETT can be misdiagnosed. To date, molecular characterization of ETTs is lacking. Furthermore, ETT is difficult to treat when disease spreads beyond the uterus. Here using RNA-Seq analysis in a cohort of ETTs and other gestational trophoblastic lesions we describe the discovery of LPCAT1-TERT fusion transcripts that occur in ETTs and coincide with underlying genomic deletions. Through cell-growth assays we demonstrate that LPCAT1-TERT fusion proteins can positively modulate cell proliferation and therefore may represent future treatment targets. Furthermore, we demonstrate that TERT upregulation appears to be a characteristic of ETTs, even in the absence of LPCAT1-TERT fusions, and that it appears linked to copy number gains of chromosome 5. No evidence of TERT upregulation was identified in other trophoblastic lesions tested, including placental site trophoblastic tumors and placental site nodules, which are thought to be the benign chorionic-type trophoblast counterpart to ETT. These findings indicate that LPCAT1-TERT fusions and copy-number driven TERT activation may represent novel markers for ETT, with the potential to improve the diagnosis, treatment, and outcome for women with this rare form of GTD