Dominique Nadine Markowski

MED12 mutations in uterine fibroids—their relationship to cytogenetic subgroups

Recurrent chromosomal alterations are found in roughly 20% of all uterine fibroids but in the majority cytogenetic changes are lacking. Recently, mutations of the gene mediator subcomplex 12 (MED12) have been detected in a majority of fibroids but no information is available whether or not they co‐occur with cytogenetic subtypes as, e.g., rearrangements of the genes encoding high mobility group AT‐hook (HMGA) proteins. In a total of 80 cytogenetically characterized fibroids from 50 patients, we were not only able to confirm the frequent occurrence of MED12 mutations but also to stratify two mutually exclusive pathways of leiomyomagenesis with either rearrangements of HMGA2 reflected by clonal chromosome abnormalities affecting 12q14∼15 or by mutations affecting exon 2 of MED12. On average the latter mutations were associated with a significantly smaller tumor size. However, G>A transitions of nucleotides c.130 or c.131 correlate with a significantly larger size of the fibroids compared to other MED12 mutations thus explaining the high prevalence of the former mutations among clinically detectable fibroids. Interestingly, fibroids with MED12 mutations expressed significantly higher levels of the gene encoding wingless‐type MMTV integration site family, member 4 (WNT4). Based on these findings and data from the literature, we hypothesize that estrogen and the mutated MED12 cooperate in activating the Wnt pathway which in turn activates β‐catenin known to cause leiomyoma‐like lesions in a mouse model. The occurrence of a “fibroid‐type mutation” in a rare histologic subtype of endometrial polyps suggests that this mechanism is not confined to uterine leiomyomas.

MED12 mutations occurring in benign and malignant mammalian smooth muscle tumors

Mutations of the mediator subcomplex 12 gene (MED12) recently have been described in a large group of uterine leiomyomas (UL) but only in a single malignant uterine smooth muscle tumor. To further address the occurrence of fibroid‐type MED12 mutations in smooth muscle tumors, we have analyzed samples from 34 leiomyosarcomas (LMS), 21 UL, two extrauterine leiomyomas (EL), and 10 canine genital leiomyomas for the presence of MED12 mutations of the UL‐type. Interestingly, besides UL MED12 mutations were found in one uterine LMS, one EL, and two canine vaginal leiomyomas. The results confirm the occurrence of fibroid‐type MED12 mutations in malignant uterine smooth muscle tumors thus suggesting a rare but existing leiomyoma‐LMS sequence. In addition, for the first time MED12 mutations are reported in smooth muscle tumors in a non‐primate mammalian species.

HMGA2 and the p19Arf-TP53-CDKN1A axis: a delicate balance in the growth of uterine leiomyomas.

Pathogenetically, uterine leiomyomas (ULs) can be interpreted as the result of a monoclonal abnormal proliferation of myometrial cells. Oncogene‐induced senescence (OIS) is a frequent phenomenon in premalignant lesions that leads to a growth arrest mainly by the activation of two potent growth‐inhibitory pathways as represented by p16Ink4a and p19Arf. The relevance of OIS for the development of UL has not been addressed, but HMGA2, encoded by a major target gene of recurrent chromosomal abnormalities in UL, has been implicated in the repression of the Ink4a/Arf (CDKN2A) locus. This prompted us to examine if HMGA2 contributes to the growth of leiomyomas by repressing this locus. Contrary to the expectations, we were able to show that generally ULs express significantly higher levels of p19Arf mRNA than myometrium and that UL with 12q14∼15 rearrangements showed higher expression levels than UL with other cytogenetic aberrations. Furthermore, the finding of a significant correlation between the expressions of p19Arf and CDKN1A shows that p19Arf triggers senescence rather than apoptosis in UL. Furthermore, the expression levels of HMGA2, p19Arf, and CDKN1A were found to be correlated with the size of the tumors, indicating that an enhanced growth potential is counterbalanced by the p19Arf pathway. Mechanistically, the UL may thus execute a program already present in their cell of origin, where it is activated to protect the genome, for example, in the case of enhanced proliferation. In summary, the results identify the p19Arf‐TP53‐CDKN1A pathway as a major player in the growth control and genomic stability of uterine fibroids.

Uterine fibroids: do we deal with more than one disease?

Uterine fibroids rank among the most frequent symptomatic human tumors at all. Recent data suggest that mutations of the mediator subcomplex 12 gene (MED12) and rearrangements of the gene-encoding high-mobility group protein AT-hook 2 (HMGA2) characterize major genetic subtypes of these tumors, which, for example, differ by their average size. Herein, we have investigated a total of 289 fibroids from 120 patients. Of these fibroids, 256 were fully genetically analyzed. Of the latter group, 20 (7.8%) fibroids had a chromosomal rearrangement of 12q14-15 reflecting a rearranged allele of HMGA2 and 179 (69.9%) fibroids had a mutation of MED12. The remaining tumors had either another genetic abnormality or no detectable abnormality at all. We were able to demonstrate that tumors of both groups also display striking differences of their frequency in individual patients. Whereas 70.0% (14/20) HMGA2-mutated fibroids made their appearance as solitary nodules, 85.5% (153/179) MED12-mutated fibroids occurred as multiple nodules as a rule of independent clonal origin, as reflected by different MED12 mutations. These findings are likely to point to a different pathogenesis of both types of fibroids. In the predominant of these groups so far, an unknown “mutator” may cause independent mutations of MED12, resulting in an independent clonal outgrowth of nodules. Furthermore, the low but existing risk of MED12-mutated fibroids to undergo malignant transformation after a leiomyoma-STUMP (smooth muscle tumors of uncertain malignant potential)-leiomyosarcoma sequence excludes the latter mutation as a suitable stand-alone marker for benign growth.

HMGA2 expression in white adipose tissue linking cellular senescence with diabetes

There is a clear link between overweight, gain of white adipose tissue, and diabetes type 2 (T2D). The molecular mechanism of the gain of adipose tissue is linked with the expression of high mobility group protein AT-hook 2 (HMGA2), and recent studies revealed an association with a SNP near HMGA2. In this study, we investigated the gene expression of HMGA2, p14Arf, CDKN1A, and BAX in human abdominal subcutaneous white adipose tissue from 157 patients. We found a significant higher HMGA2 expression in obese individuals than in non-obese patients. Furthermore, the HMGA2 expression in white adipose tissue in patient with type 2 diabetes was significantly higher than in nondiabetic patients. There is an association between the DNA-binding nonhistone protein HMGA2 and the risk of developing T2D that remains mechanistically unexplained so far. Likewise, p14Arf, an inducer of cellular senescence, has been associated with the occurrence of T2D. The data of the present study provide evidence that both proteins act within the same network to drive proliferation of adipose tissue stem and precursor cells, senescence, and increased risk of T2D, respectively.

p14Arf acts as an antagonist of HMGA2 in senescence of mesenchymal stem cells—implications for benign tumorigenesis

HMGA2 is a major regulator of benign tumorigenesis from mesenchyme‐derived tissues and stem‐cell self‐renewal. It has been postulated that HMGA2 mediates its critical function by decreasing p16Ink4a/p14Arf expression and cellular senescence. To repress the oncogenic activity of HMGA2, the lin‐28‐let‐7 axis is thought to increasingly repress the expression of HMGA2 with age. To understand the HMGA2‐p14Arf‐relationship in benign tumorigenesis, we performed a series of experiments on mesenchymal stem‐cells, i.e., the proposed cells of origin of lipomas and uterine leiomyomas. The expression of both genes was inversely correlated during senescence in vitro but contrary to the expectations in adipose tissue derived stem cells (ADSCs) stimulation of HMGA2 by FGF1 increased the expression of p14Arf. Based on the assumption that in ADSCs p14Arf is repressing HMGA2, siRNA silencing of p14Arf was performed resulting in a significant upregulation of HMGA2. To see if p14Arf can repress HMGA2 by a TP53‐dependent mechanism, nutlin‐3, a known MDM2 antagonist, was used which not only increased the activity of the senescence, associated markers p21 and beta‐galactosidase, but also decreased the expression of HMGA2, suggesting that p14Arf indeed influences HMGA2 by a p53‐dependent mechanism because nutlin‐3 stabilizes p53. Accordingly, the HMGA2 response triggered by serum was reduced by treatment of ADSCs with nutlin‐3. As to the interaction between HMGA2 and p14Arf in benign tumorigenesis, we propose a model where akin to MSC self‐renewal during tissue repair the simultaneous increase of p14Arf with HMGA2 ensures genomic stability, whereas in turn p14Arf can repress HMGA2 via TP53.

Cell culture and senescence in uterine fibroids.

The in vitro growth of cells from uterine fibroids is characterized by an early onset of senescence. Often, an even lower growth potential than that of matching myometrial cells is noted. Also, the tremendous differences in the expression of the high mobility group protein HMGA2 seen when comparing fibroids of different genetic subtypes are surprisingly not reflected by significant differences in their growth potential in vitro. We aimed to evaluate possible changes of the HMGA2 expression level between the native tissue and cell cultures, so we performed quantitative real-time polymerase chain reaction studies that revealed a marked decrease of the HMGA2 mRNA in culture in those cases with overexpression of HMGA2. In the two cases initially showing the highest expression, it decreased by approximately 97%. Associated with the decrease of HMGA2 was a clearly increased expression of the senescence-associated p19(Arf). Together, these findings explain the similar behavior of cell cultures from fibroids of different genetic subgroups and may also offer an explanation for the early onset of in vitro senescence in these cell cultures.

Selective visual attention ensures constancy of sensory representations: Testing the influence of perceptual load and spatial competition

We report findings from several variants of a psychophysical experiment using an acceleration detection task in which we tested predictions derived from recent neurophysiological data obtained from monkey area MT. The task was designed as a Posner paradigm and required subjects to detect the speed-up of a moving bar, cued with 75% validity. Displays varied according to number of simultaneously presented objects, spatial distance, and difficulty of the task. All data obtained under different levels of competition with multiple objects were compared to a corresponding condition, in which only a single moving bar was presented in the absence of any interfering distracter object. For attended objects, subjects did not show any difference in their ability to detect accelerations, regardless of the strength of inter-object competition or spatial distance. This finding was consistent in all of the experiments, and was even obtained when the acceleration was made hardly detectable. In contrast, increasing competitive interactions either by enhancing number of objects or spatial proximity resulted in strong reduction of performance for non-attended objects. The findings support current noise reduction models and suggest that attention adjusts neuronal processing to ensure a constant sensory representation of the attended object as if this object was the only one in the scene.

Correlated Expression of HMGA2 and PLAG1 in Thyroid Tumors, Uterine Leiomyomas and Experimental Models

In pleomorphic adenomas of the salivary glands (PASG) recurrent chromosomal rearrangements affecting either 8q12 or 12q14∼15 lead to an overexpression of the genes of the genuine transcription factor PLAG1 or the architectural transcription factor HMGA2, respectively. Both genes are also affected by recurrent chromosomal rearrangements in benign adipocytic tumors as e. g. lipomas and lipoblastomas. Herein, we observed a strong correlation between the expression of HMGA2 and PLAG1 in 14 benign and 23 malignant thyroid tumors. To address the question if PLAG1 can be activated by HMGA2, the expression of both genes was quantified in 32 uterine leiomyomas 17 of which exhibited an overexpression of HMGA2. All leiomyomas with HMGA2 overexpression also revealed an activation of PLAG1 in the absence of detectable chromosome 8 abnormalities affecting the PLAG1 locus. To further investigate if the overexpression of PLAG1 is inducible by HMGA2 alone, HMGA2 was transiently overexpressed in MCF-7 cells. An increased PLAG1 expression was observed 24 and 48 h after transfection. Likewise, stimulation of HMGA2 by FGF1 in adipose tissue-derived stem cells led to a simultaneous increase of PLAG1 mRNA. Altogether, these data suggest that HMGA2 is an upstream activator of PLAG1. Accordingly, this may explain the formation of tumors as similar as lipomas and lipoblastomas resulting from an activation of either of both genes by chromosomal rearrangements.

Molecular topography of the MED12-deleted region in smooth muscle tumors: a possible link between non-B DNA structures and hypermutability

BackgroundDeletions of the gene encoding mediator subcomplex 12 (MED12) in human smooth muscle tumors rank among the most frequent genomic alterations in human tumors at all. In a minority of these cases, small deletions are found. In an attempt to delineate key features of the deletions aimed at a better understanding of the molecular pathogenesis of uterine smooth muscle tumors we have analyzed 70 MED12 deletions including 46 cases from the literature and 24 own unpublished cases.ResultsThe average length of the deletions was 18.7 bp ranging between 2 bp and 43 bp. While in general multitudes of 3 clearly dominated leaving the transcript in frame, deletions of 21, 24, 30, and 33 nucleotides were clearly underrepresented. Within the DNA segment affected deletion breakpoints were not randomly distributed. Most breakpoints clustered within the center of the segment where two peaks of breakpoint clusters could be distinguished. Interestingly, one of these clusters coincides with the loop of a putative folded non-B DNA structure whereas a much lower number of breaks noted in the 5′ and 3′ stem of the structure forming an intramolecular B-helix. The second cluster mainly consisting of 3′ breaks was located in a region downstream adjacent to the stem.ConclusionThe present study describes for the first time main characteristics of MED12 deletions occurring in smooth muscle tumors. Interestingly, the non-random distribution of breakpoints within the deletion hotspot region may point to a role of non-canonical DNA structures for the occurrence of these mutations and the molecular pathogenesis of uterine smooth muscle tumors, respectively.