BackgroundUterine leiomyoma is the most common benign tumor in reproductive-age women. Each leiomyoma is thought to be a benign monoclonal tumor arising from a single transformed myometrial smooth muscle cell; however, it is not known what leiomyoma cell type is responsible for tumor growth. Thus, we tested the hypothesis that a distinct stem/reservoir cell-enriched population, designated as the leiomyoma-derived side population (LMSP), is responsible for cell proliferation and tumor growth.Principal FindingsLMSP comprised approximately 1% of all leiomyoma and 2% of all myometrium-derived cells. All LMSP and leiomyoma-derived main population (LMMP) but none of the side or main population cells isolated from adjacent myometrium carried a mediator complex subunit 12 mutation, a genetic marker of neoplastic transformation. Messenger RNA levels for estrogen receptor-α, progesterone receptor and smooth muscle cell markers were barely detectable and significantly lower in the LMSP compared with the LMMP. LMSP alone did not attach or survive in monolayer culture in the presence or absence of estradiol and progestin, whereas LMMP readily grew under these conditions. LMSP did attach and survive when directly mixed with unsorted myometrial cells in monolayer culture. After resorting and reculturing, LMSP gained full potential of proliferation. Intriguingly, xenografts comprised of LMSP and unsorted myometrial smooth muscle cells grew into relatively large tumors (3.67±1.07 mm3), whereas xenografts comprised of LMMP and unsorted myometrial smooth muscle cells produced smaller tumors (0.54±0.20 mm3, p<0.05, n = 10 paired patient samples). LMSP xenografts displayed significantly higher proliferative activity compared with LMMP xenografts (p<0.05).ConclusionsOur data suggest that LMSP, which have stem/reservoir cell characteristics, are necessary for in vivo growth of leiomyoma xenograft tumors. Lower estrogen and progesterone receptor levels in LMSP suggests an indirect paracrine effect of steroid hormones on stem cells via the mature neighboring cells.

Uterine leiomyomas are extremely common estrogen and progesterone-dependent tumors of the myometrium and cause irregular uterine bleeding, severe anemia, and recurrent pregnancy loss in 15-30% of reproductive-age women. Each leiomyoma is thought to arise from a single mutated myometrial smooth muscle stem cell. Leiomyoma side-population (LMSP) cells comprising 1% of all tumor cells and displaying tumor-initiating stem cell characteristics are essential for estrogen-and progesterone-dependent in vivo growth of tumors, although they have remarkably lower estrogen/progesterone receptor levels than mature myometrial or leiomyoma cells. However, how estrogen/progesterone regulates the growth of LMSP cells via mature neighboring cells is unknown. Here, we demonstrate a critical paracrine role of the wingless-type (WNT)/β-catenin pathway in estrogen/progesterone-dependent tumorigenesis, involving LMSP and differentiated myometrial or leiomyoma cells. Estrogen/progesterone treatment of mature myometrial cells induced expression of WNT11 and WNT16, which remained constitutively elevated in leiomyoma tissues. In LMSP cells cocultured with mature myometrial cells, estrogen-progesterone selectively induced nuclear translocation of β-catenin and induced transcriptional activity of its heterodimeric partner T-cell factor and their target gene AXIN2, leading to the proliferation of LMSP cells. This effect could be blocked by a WNT antagonist. Ectopic expression of inhibitor of β-catenin and T-cell factor 4 in LMSP cells, but not in mature leiomyoma cells, blocked the estrogen/ progesterone-dependent growth of human tumors in vivo. We uncovered a paracrine role of the WNT/β-catenin pathway that enables mature myometrial or leiomyoma cells to send mitogenic signals to neighboring tissue stem cells in response to estrogen and progesterone, leading to the growth of uterine leiomyomas.WNT/β-catenin signaling | paracrine signaling | tumor biology

Recent identification of somatic MED12 mutations in most uterine leiomyomas brings a new venue for the study of the tumorigenesis of leiomyomas. We are particularly interested in the correlation of MED12 and HMGA2 gene products in leiomyomas and leiomyosarcomas with and without MED12 mutations. To address these issues, in this study we examined MED12 mutations in a large cohort of usual type leiomyomas (178 cases) and uterine leiomyosarcomas (32 cases). We found that 74.7% (133/178) of leiomyomas had MED12 mutations, which was consistent with several independent studies. In contrast, only 9.7% (3/32) of leiomyosarcomas harbored MED12 mutations. Expression analysis by Western blot and immunohistochemistry revealed that those leiomyomas with complex MED12 mutations had significantly lower protein products than matched myometrium. Interestingly, most leiomyosarcomas without MED12 mutations also had very low levels of MED12 expression in comparison to the matched myometrium. These findings suggest a potential functional role of MED12 in both benign and malignant uterine smooth muscle tumors. When we further examined HMGA2 expression in all leiomyomas and leiomyosarcomas, we found HMGA2 overexpression was exclusively present in those leiomyomas with no MED12 mutation, accounting for 10.1 % (18/178) of total leiomyomas and 40 % (18/45) of non-MED12 mutant leiomyomas. Twenty-five % (8/32) of leiomyosarcomas had HMGA2 overexpression and no MED12 mutations were found in HMGA2 positive leiomyosarcoma. These findings strongly suggest that MED12 mutations and HMGA2 overexpression are independent genetic events that occur in leiomyomas, and they may act differently in the tumorigenesis of uterine leiomyomas.

Patient-derived pancreatic ductal adenocarcinoma (PDAC) organoid systems show great promise for understanding the biological underpinnings of disease and advancing therapeutic precision medicine. Despite the increased use of organoids, the fidelity of molecular features, genetic heterogeneity, and drug response to the tumor of origin remain important unanswered questions limiting their utility. To address this gap in knowledge, primary tumor- and patient-derived xenograft (PDX)-derived organoids, and 2D cultures for in-depth genomic and histopathologic comparisons with the primary tumor were created. Histopathologic features and PDAC representative protein markers (e.g., claudin 4 and CA19-9) showed strong concordance. DNA- and RNA-sequencing (RNAseq) of single organoids revealed patient-specific genomic and transcriptomic consistency. Single-cell RNAseq demonstrated that organoids are primarily a clonal population. In drug response assays, organoids displayed patient-specific sensitivities. In addition, the PDX response to FOLFIRINOX and gemcitabine/abraxane treatments were examined, which was recapitulated with organoids. This study has demonstrated that organoids are potentially invaluable for precision medicine as well as preclinical drug treatment studies because they maintain distinct patient phenotypes and respond differently to drug combinations and dosage. The patient-specific molecular and histopathologic fidelity of organoids indicate that they can be used to understand the etiology of the patient's tumor and the differential response to therapies and suggests utility for predicting drug responses.

The neuroimmunodegenerative syndrome that develops in mice infected with ts1, a mutant of Moloney murine leukemia virus, resembles human AIDS. Both ts1 and human immunodeficiency virus type 1 infect astrocytes, microglia, and oligodendrocytes but do not infect neurons. Oxidative stress has been implicated in the neuropathology of AIDS dementia and other neurodegenerative diseases. We report here that ts1 infection of astrocytes (both transformed C1 cells and primary cultures) also induces thiol (i.e., glutathione and cysteine) depletion and reactive oxygen species (ROS) accumulation, events occurring in parallel with viral envelope precursor gPr80(env) accumulation and upregulated expression of endoplasmic reticulum chaperones GRP78 and GRP94. Furthermore, ts1-infected astrocytes mobilize their thiol redox defenses by upregulating levels of the Nrf-2 transcription factor, as well its targets, the xCT cystine/glutamate antiporter, gamma-glutamylcysteine ligase, and glutathione peroxidase. Depleting intracellular thiols by treating uninfected astrocytes with buthionine sulfoximine (BSO), a glutathione synthesis inhibitor, or by culturing in cystine-deficient medium, also induces ROS accumulation, activates Nrf-2, and upregulates Nrf-2 target gene expression in these astrocytes. Overexpression of Nrf-2 in astrocytes specifically increases expression of the above thiol synthesis-related proteins. Further treatment with BSO or N-acetylcysteine in transfected cells modulates this expression. Thiol depletion also accelerates cell death, while thiol supplementation promotes survival of ts1-infected cells. Together, our results indicate that ts1 infection of astrocytes, along with ts1-induced gPr80(env) accumulation, endoplasmic reticulum stress, thiol depletion, and oxidative stress, accelerates cell death; in response to the thiol depletion and oxidative stress, astrocytes activate their Nrf-2-mediated thiol antioxidant defenses, promoting cell survival.