HMGA2 overexpression induces plasticity in myometrial cells and a transcriptomic profile more similar to that of uterine fibroids.

OBJECTIVE: To study the possible role for HMGA2 overexpression in differentiated myometrial cells and its potential to induce a stem cell-like or dedifferentiating phenotype and drive fibroid development. DESIGN: Myometrial cells were immortalized and transduced with an HMGA2 lentivirus to produce HMGA2hi cells. In vitro stem cell assays were conducted and RNA from HMGA2hi and control cells and fibroid-free myometrial (MyoN) and HMGA2 fibroid (HMGA2F) tissues were submitted for RNA-sequencing. SETTING: University research laboratory SUBJECTS: Women undergoing hysterectomy for symptomatic uterine fibroids or other gynecological conditions. INTERVENTION: Not applicable. MAIN OUTCOME MEASURES: In-vitro stem-like properties from myometrium cell lines. RNA-sequencing and collagen production of HMGA2 overexpressing primary leiomyoma tissue and cell lines. RESULTS: HMGA2hi cells have enhanced self-renewal capacity, decreased proliferation, and have a greater ability to differentiate into other mesenchymal cell types. HMGA2hi cells exhibit a stem cell-like signature and share transcriptomic similarities with HMGA2F. Moreover, dysregulated extracellular matrix pathways are observed in both HMGA2hi cells and HMGA2F. CONCLUSION: Our findings suggest that HMGA2 overexpression drives myometrial cells to dedifferentiate into a more plastic phenotype and provides evidence for an alternative mechanism for fibroid etiology, suggesting that fibroids may not only arise from a mutated stem cell but also from a mutated differentiated myometrial cell.

Unraveling the Molecular Landscape of Uterine Fibroids, Insights into HMGA2 and Stem Cell Involvement.

Uterine fibroids are prevalent benign tumors in women that exhibit considerable heterogeneity in clinical presentation and molecular characteristics, necessitating a deeper understanding of their etiology and pathogenesis. HMGA2 overexpression has been associated with fibroid development, yet its precise role remains elusive. Mutations in fibroids are mutually exclusive and largely clonal, suggesting that tumors originate from a single mutant cell. We explored a possible role for HMGA2 overexpression in differentiated myometrial cells, hypothesizing its potential to induce a stem cell-like or dedifferentiating phenotype and drive fibroid development. Myometrial cells were immortalized and transduced with an HMGA2 lentivirus to produce HMGA2hi cells. In vitro stem cell assays were conducted and RNA from HMGA2hi and control cells and fibroid-free myometrial and HMGA2 fibroid (HMGA2F) tissues were submitted for RNA-sequencing. HMGA2hi cells have enhanced self-renewal capacity, decreased proliferation, and have a greater ability to differentiate into other mesenchymal cell types. HMGA2hi cells exhibit a stem cell-like signature and share transcriptomic similarities with HMGA2F. Moreover, dysregulated extracellular matrix pathways are observed in both HMGA2hi cells and HMGA2F. Our findings suggest that HMGA2 overexpression drives myometrial cells to dedifferentiate into a more plastic phenotype and underscore a pivotal role for HMGA2 in fibroid pathogenesis.

Cysteine-rich intestinal protein 1 is a novel surface marker for human myometrial stem/progenitor cells.

Myometrial stem/progenitor cells (MyoSPCs) have been proposed as the cells of origin for uterine fibroids, but the identity of the MyoSPC has not been well established. We previously identified SUSD2 as a possible MyoSPC marker, but the relatively poor enrichment in stem cell characteristics of SUSD2+ over SUSD2- cells compelled us to find better markers. We combined bulk RNA-seq of SUSD2+/- cells with single cell RNA-seq to identify markers for MyoSPCs. We observed seven distinct cell clusters within the myometrium, with the vascular myocyte cluster most highly enriched for MyoSPC characteristics and markers. CRIP1 expression was found highly upregulated by both techniques and was used as a marker to sort CRIP1+/PECAM1- cells that were both enriched for colony forming potential and able to differentiate into mesenchymal lineages, suggesting that CRIP1+/PECAM1- cells could be used to better study the etiology of uterine fibroids.

Cysteine-Rich Intestinal Protein 1 is a Novel Surface Marker for Myometrial Stem/Progenitor Cells.

Myometrial stem/progenitor cells (MyoSPCs) have been proposed as the cells of origin for uterine fibroids, which are benign tumors that develop in the myometrium of most reproductive age women, but the identity of the MyoSPC has not been well established. We previously identified SUSD2 as a possible MyoSPC marker, but the relatively poor enrichment in stem cell characteristics of SUSD2+ over SUSD2- cells compelled us to find better discerning markers for more rigorous downstream analyses. We combined bulk RNA-seq of SUSD2+/- cells with single cell RNA-seq to identify markers capable of further enriching for MyoSPCs. We observed seven distinct cell clusters within the myometrium, with the vascular myocyte cluster most highly enriched for MyoSPC characteristics and markers, including SUSD2. CRIP1 expression was found highly upregulated in both techniques and was used as a marker to sort CRIP1+/PECAM1- cells that were both enriched for colony forming potential and able to differentiate into mesenchymal lineages, suggesting that CRIP1+/PECAM1- cells could be used to better study the etiology of uterine fibroids.

Transcriptome and DNA methylome analyses reveal underlying mechanisms for the racial disparity in uterine fibroids.

Uterine fibroids (leiomyomas) affect Black women disproportionately compared with women of other races and ethnicities in terms of prevalence, incidence, and severity of symptoms. The causes of this racial disparity are essentially unknown. We hypothesized that myometria of Black women are more susceptible to developing fibroids, and we examined the transcriptomic and DNA methylation profiles of myometria and fibroids from Black and White women for comparison. Myometrial samples cluster by race in both their transcriptome and DNA methylation profiles, whereas fibroid samples only cluster by race in the latter. More differentially expressed genes (DEGs) were detected in the Black and White myometrial sample comparison than in the fibroid comparison. Leiomyoma gene set expression analysis identified 4 clusters of DEGs, including a cluster of 24 genes with higher expression in myometrial samples from Black women. One of the DEGs in this group, von Willibrands factor (VWF), was significantly hypomethylated in both myometrial samples from Black women and in all fibroids at 2 CpG probes that are near a putative enhancer site and that are correlated with VWF expression levels. These results suggest that the molecular basis for the disparity in fibroid disease between Black and White women could be found in the myometria before fibroid development and not in the fibroids themselves.

Effect of Cloth Masks and N95 Respirators on Maximal Exercise Performance in Collegiate Athletes.

This study compared exercise performance and comfort while wearing an N95 filtering facepiece respirator (N95), cloth mask, or no intervention control for source control during a maximal graded treadmill exercise test (GXT). Twelve Division 1 athletes (50% female, age = 20.1 ± 1.2, BMI = 23.5 ± 1.6) completed GXTs under three randomized conditions (N95, cloth mask, control). GXT duration, heart rate (HR), respiration rate (RR), transcutaneous oxygen saturation (SpO2), transcutaneous carbon dioxide (TcPCO2), rating of perceived exertion (RPE), and perceived comfort were measured. Participants ran significantly longer in control (26.06 min) versus N95 (24.20 min, p = 0.03) or cloth masks (24.06 min, p = 0.04). No differences occurred in the slope of HR or SpO2 across conditions (p > 0.05). TcPCO2 decreased faster in control (B = −0.89) versus N95 (B = 0.14, p = 0.02) or cloth masks (B = −0.26, p = 0.03). RR increased faster in control (B = 8.32) versus cloth masks (B = 6.20, p = 0.04). RPE increased faster in the N95 (B = 1.91) and cloth masks (B = 1.79) versus control (B = 1.59, p < 0.001 and p = 0.05, respectively). Facial irritation/itching/pinching was higher in the N95 versus cloth masks, but sweat/moisture buildup was lower (p < 0.05 for all). Wearing cloth masks or N95s for source control may impact exercise performance, especially at higher intensities. Significant physiological differences were observed between cloth masks and N95s compared to control, while no physiological differences were found between cloth masks and N95s; however, comfort my differ.

Development of a 17-Plex of Penta- and Tetra-Nucleotide Microsatellites for DNA Profiling and Paternity Testing in Horses.

Tetranucleotide and pentanucleotide short tandem repeat (hereafter termed tetraSTR and pentaSTR) polymorphisms have properties that make them desirable for DNA profiling and paternity testing. However, certain species, such as the horse, have far fewer tetraSTRs than other species and for this reason dinucleotide STRs (diSTRs) have become the standard for DNA profiling in horses, despite being less desirable for technical reasons. During our testing of a series of candidate genes as potentially underlying a heritable condition characterized by megaesophagus in the Friesian horse breed, we found that good tetraSTRs do exist in horses but, as expected, at a much lower frequency than in other species, e.g., dogs and humans. Using a series of efficient methods developed in our laboratory for the production of multiplexed tetraSTRs in other species, we identified a set of tetra- and pentaSTRs that we developed into a 17-plex panel for the horse, plus a sex-identifying marker near the amelogenin gene. These markers were tested in 128 horses representing 16 breeds as well as crossbred horses, and we found that these markers have useful genetic variability. Average observed heterozygosities (Ho) ranged from 0.53 to 0.89 for the individual markers (0.66 average Ho for all markers), and 0.62-0.82 for expected heterozygosity (He) within breeds (0.72 average He for all markers). The probability of identity (PI) within breeds for which 10 or more samples were available was at least 1.1 x 10-11, and the PI among siblings (PIsib) was 1.5 x 10-5. Stutter was ≤ 11% (average stutter for all markers combined was 6.9%) compared to the more than 30% typically seen with diSTRs. We predict that it will be possible to develop accurate allelic ladders for this multiplex panel that will make cross-laboratory comparisons easier and will also improve DNA profiling accuracy. Although we were only able to exclude candidate genes for Friesian horse megaesophagus with no unexcluded genes that are possibly causative at this point in time, the study helped us to refine the methods used to develop better tetraSTR multiplexed panels for species such as the horse that have a low frequency of tetraSTRs.

Transcriptome Analyses of Myometrium from Fibroid Patients Reveals Phenotypic Differences Compared to Non-Diseased Myometrium.

Uterine fibroid tissues are often compared to their matched myometrium in an effort to understand their pathophysiology, but it is not clear whether the myometria of uterine fibroid patients represent truly non-disease control tissues. We analyzed the transcriptomes of myometrial samples from non-fibroid patients (M) and compared them with fibroid (F) and matched myometrial (MF) samples to determine whether there is a phenotypic difference between fibroid and non-fibroid myometria. Multidimensional scaling plots revealed that M samples clustered separately from both MF and F samples. A total of 1169 differentially expressed genes (DEGs) (false discovery rate < 0.05) were observed in the MF comparison with M. Overrepresented Gene Ontology terms showed a high concordance of upregulated gene sets in MF compared to M, particularly extracellular matrix and structure organization. Gene set enrichment analyses showed that the leading-edge genes from the TGFß signaling and inflammatory response gene sets were significantly enriched in MF. Overall comparison of the three tissues by three-dimensional principal component analyses showed that M, MF, and F samples clustered separately from each other and that a total of 732 DEGs from F vs. M were not found in the F vs. MF, which are likely understudied in the pathogenesis of uterine fibroids and could be key genes for future investigation. These results suggest that the transcriptome of fibroid-associated myometrium is different from that of non-diseased myometrium and that fibroid studies should consider using both matched myometrium and non-diseased myometrium as controls.

In Vivo Cell Fate Tracing Provides No Evidence for Mesenchymal to Epithelial Transition in Adult Fallopian Tube and Uterus.

The mesenchymal to epithelial transition (MET) is thought to be involved in the maintenance, repair, and carcinogenesis of the fallopian tube (oviduct) and uterine epithelium. However, conclusive evidence for the conversion of mesenchymal cells to epithelial cells in these organs is lacking. Using embryonal cell lineage tracing with reporters driven by mesenchymal cell marker genes of the female reproductive tract (AMHR2, CSPG4, and PDGFRß), we show that these reporters are also expressed by some oviductal and uterine epithelial cells at birth. These mesenchymal reporter-positive epithelial cells are maintained in adult mice across multiple pregnancies, respond to ovarian hormones, and form organoids. However, no labeled epithelial cells are present in any oviductal or uterine epithelia when mesenchymal cell labeling was induced in adult mice. Organoids developed from mice labeled in adulthood were also negative for mesenchymal reporters. Collectively, our work found no definitive evidence of MET in the adult fallopian tube and uterine epithelium.

Putative human myometrial and fibroid stem-like cells have mesenchymal stem cell and endometrial stromal cell properties.

STUDY QUESTION: Can endometrial stromal stem/progenitor cell markers, SUSD2 and CD146/CD140b, enrich for human myometrial and fibroid stem/progenitor cells? SUMMARY ANSWER: SUSD2 enriches for myometrial and fibroid cells that have mesenchymal stem cell (MSC) characteristics and can also be induced to decidualise. WHAT IS KNOWN ALREADY: Mesenchymal stem-like cells have been separately characterised in the endometrial stroma and myometrium and may contribute to diseases in their respective tissues. STUDY DESIGN, SIZE, DURATION: Normal myometrium, fibroids and endometrium were collected from hysterectomies with informed consent. Primary cells or tissues were used from at least three patient samples for each experiment. PARTICIPANTS/MATERIALS, SETTING, METHODS: Flow cytometry, immunohistochemistry and immunofluorescence were used to characterise tissues. In vitro colony formation in normoxic and hypoxic conditions, MSC lineage differentiation (osteogenic and adipogenic) and decidualisation were used to assess stem cell activity. Xenotransplantation into immunocompromised mice was used to determine in vivo stem-like activity. Endpoint measures included quantitative PCR, colony formation, trichrome, Oil Red O and alkaline phosphatase activity staining. MAIN RESULTS AND THE ROLE OF CHANCE: CD146+CD140b+ and/or SUSD2+ myometrial and fibroid cells were located in the perivascular region and formed more colonies in vitro compared to control cells and differentiated down adipogenic and osteogenic mesenchymal lineages in vitro. SUSD2+ myometrial cells had greater in vitro decidualisation potential, and SUSD2+ fibroid cells formed larger tumours in vivo compared to control cells. LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Markers used in this study enrich for cells with stem/progenitor cell activity; however, they do not distinguish stem from progenitor cells. SUSD2+ myometrial cells express markers of decidualisation when treated in vitro, but in vivo assays are needed to fully demonstration their ability to decidualise. WIDER IMPLICATIONS OF THE FINDINGS: These results suggest a possible common MSC for the endometrial stroma and myometrium, which could be the tumour-initiating cell for uterine fibroids. STUDY FUNDING/COMPETING INTEREST(S): These studies were supported by NIH grants to JMT (R01OD012206) and to ALP (F32HD081856). The authors certify that we have no conflicts of interest to disclose.

Integrated Epigenome, Exome, and Transcriptome Analyses Reveal Molecular Subtypes and Homeotic Transformation in Uterine Fibroids.

Uterine fibroids are benign myometrial smooth muscle tumors of unknown etiology that, when symptomatic, are the most common indication for hysterectomy in the United States. Unsupervised clustering of results from DNA methylation analyses segregates normal myometrium from fibroids and further segregates the fibroids into subtypes characterized by MED12 mutation or activation of either HMGA2 or HMGA1 expression. Upregulation of HMGA2 expression does not always appear to be dependent on translocation but is associated with hypomethylation in the HMGA2 gene body. HOXA13 expression is upregulated in fibroids and correlates with expression of typical uterine fibroid genes. Significant overlap of differentially expressed genes is observed between cervical stroma and uterine fibroids compared with normal myometrium. These analyses show a possible role of DNA methylation in fibroid biology and suggest that homeotic transformation of myometrial cells to a more cervical stroma phenotype could be an important mechanism for etiology of the disease.

Label-Retaining, Putative Mesenchymal Stem Cells Contribute to Murine Myometrial Repair During Uterine Involution.

Uterine remodeling during pregnancy is a fundamental, dynamic process required for successful propagation of eutherian species. The uterus can increase in size up to 40-fold during pregnancy, which is largely attributed to expansion of the myometrium by hyperplasia and hypertrophy. After pregnancy, the uterus repairs the remodeled or "damaged" tissue during uterine involution (INV). Little is known about this repair process, particularly the role of mesenchymal stem/progenitor cells. The objective of this study was to identify and characterize putative mesenchymal stem/progenitor cells in the murine myometrium using a combination of label retention and mesenchymal stem cell (MSC) marker expression and a pregnancy and uterine INV model. Tet-off transgenic mice with the Cre-lox system were used to specifically label mesenchymal cells (ie, myometrial and endometrial stromal cells) within the uterus while avoiding other cell types (eg, epithelial, immune, and endothelial cells) to identify slowly dividing cells and assess their stem cell qualities. We identified myometrial label-retaining cells (LRCs) that persisted for at least 3 months, expressed CD146 and CD140b (MSC markers), and proliferated at a higher rate during uterine INV compared with nonlabeled cells. The LRCs did not appear to express either estrogen receptor alpha or progesterone receptor, nor did the number of LRCs change at different estrous stages or in response to exogenous estradiol or progesterone administration, suggesting that LRCs were not involved in normal estrous cycling. The results from this study provide important insight into putative stem/progenitor cells in the myometrium and their possible role in uterine physiology.

Nuclear PTEN Localization Contributes to DNA Damage Response in Endometrial Adenocarcinoma and Could Have a Diagnostic Benefit for Therapeutic Management of the Disease.

Endometrial adenocarcinoma (EndoCA) is the most common gynecologic cancer type in the United States, and its incidence is increasing. The majority of patients are disease-free after surgical resection of stage I tumors, which is often followed by radiotherapy, but most patients with advanced disease recur and have a poor prognosis, largely because the tumors become refractory to cytotoxic chemotherapies. PTEN, a commonly mutated tumor suppressor in EndoCAs, is well known for its ability to inhibit the AKT/mTOR signaling pathway. Nuclear functions for PTEN have been proposed as well, but whether those affect EndoCA development, progression, or outcomes is not well understood. Using immunohistochemistry, nuclear PTEN expression was observed in approximately half of EndoCA patient tumors, independent of grade and cytoplasmic PTEN expression. Higher levels of the DNA damage response (DDR) marker, γH2AX, were observed by immunohistochemistry and immunofluorescence in human EndoCA tumor sections that were PTEN-negative, in murine EndoCA tissues that were genetically modified to be PTEN-null, and in Ishikawa EndoCA cells, which do not express endogenous PTEN. Overexpression of exogenous PTEN-WT or PTEN-NLS, a modified PTEN with an added nuclear localization signal, significantly improved both DDR and G2-M transition in Ishikawa cells treated with a DNA-damaging agent. Whereas PARP inhibition with Olaparib was not as effective in Ishikawa cells expressing native or PTEN-NLS, inhibition with Talazoparib was not affected by PTEN overexpression. These results suggest that nuclear PTEN subcellular localization in human EndoCA could be diagnostic when considering DDR therapeutic intervention. Mol Cancer Ther; 17(9); 1995-2003. ©2018 AACR.