Kenji Unno

Role of Stem Cells in Human Uterine Leiomyoma Growth

BACKGROUND Uterine 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 FINDINGS LMSP 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 mm(3)), whereas xenografts comprised of LMMP and unsorted myometrial smooth muscle cells produced smaller tumors (0.54 ± 0.20 mm(3), p<0.05, n = 10 paired patient samples). LMSP xenografts displayed significantly higher proliferative activity compared with LMMP xenografts (p<0.05). CONCLUSIONS Our 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.

The Development of Cervical and Vaginal Adenosis as a Result of Diethylstilbestrol Exposure In Utero

Exposure to exogenous hormones during development can result in permanent health problems. In utero exposure to diethylstilbestrol (DES) is probably the most well documented case in human history. DES, an orally active synthetic estrogen, was believed to prevent adverse pregnancy outcome and thus was routinely given to selected pregnant women from the 1940s to the 1960s. It has been estimated that 5 million pregnant women worldwide were prescribed DES during this period. In the early 1970s, vaginal clear cell adenocarcinomas (CCACs) were diagnosed in daughters whose mother took DES during pregnancy (known as DES daughters). Follow-up studies demonstrated that exposure to DES in utero causes a spectrum of congenital anomalies in female reproductive tracts and CCACs. Among those, cervical and vaginal adenoses are most commonly found, which are believed to be the precursors of CCACs. Transformation related protein 63 (TRP63/p63) marks the cell fate decision of Müllerian duct epithelium (MDE) to become squamous epithelium in the cervix and vagina. DES disrupts the TRP63 expression in mice and induces adenosis lesions in the cervix and vagina. This review describes mouse models that can be used to study the development of DES-induced anomalies, focusing on cervical and vaginal adenoses, and discusses their molecular pathogenesis.

Increased AKT or MEK1/2 Activity Influences Progesterone Receptor Levels and Localization in Endometriosis

CONTEXT Endometriosis is characterized by progesterone resistance and hyperactivity of the AKT and MAPK pathways. Kinases can cause posttranslational modifications of the progesterone receptor (PR) to influence cellular localization and protein stability. OBJECTIVE The objective of this study was to determine whether the increased AKT or MAPK kinase-1/2 (MEK1/2) activity observed in endometriotic stromal cells (OSIS) from ovarian endometriomas influences levels of PR protein. In turn, the effects of inhibiting AKT or MEK1/2 in the presence of the progestin R5020 on cell viability were investigated. RESULTS Inhibiting AKT with MK-2206 or MEK1/2 with U0126 for 24 hours in the absence of R5020 increased total and nuclear PRA and PRB protein levels in OSIS but not in eutopic endometrial stromal cells from disease-free patients from disease-free patients. MK-2206 and R5020 decreased OSIS viability and increased apoptosis. Trends toward decreased volumes of sc grafted endometriosis tissues were demonstrated with MK-2206 and progesterone. CONCLUSIONS Inhibition of AKT or MEK1/2 increased total and nuclear PR protein in OSIS. MK-2206 and R5020 decreased OSIS viability and increased apoptosis. The AKT and MAPK pathways may be potential molecular targets for the treatment of endometriosis.

Bmi1 marks distinct castration-resistant luminal progenitor cells competent for prostate regeneration and tumour initiation.

Identification of defined cell populations with stem/progenitor properties is key for understanding prostate development and tumorigenesis. Here we show that the polycomb repressor protein Bmi1 marks a population of castration-resistant luminal epithelial cells enriched in the mouse proximal prostate. We employ lineage tracing to show that these castration-resistant Bmi1-expressing cells (or CARBs) are capable of tissue regeneration and self-renewal. Notably, CARBs are distinct from the previously described luminal castration-resistant Nkx3.1-expressing cells (CARNs). CARBs can serve as a prostate cancer cell-of-origin upon Pten deletion, yielding luminal prostate tumours. Clonal analysis using the R26R-confetti allele indicates preferential tumour initiation from CARBs localized to the proximal prostate. These studies identify Bmi1 as a marker for a distinct population of castration-resistant luminal epithelial cells enriched in the proximal prostate that can serve as a cell of origin for prostate cancer.

Establishment of Human Patient-Derived Endometrial Cancer Xenografts in NOD scid Gamma Mice for the Study of Invasion and Metastasis

Objective Most endometrial cancers are detected early and have a good prognosis, while some endometrial cancers are highly invasive, metastasize early, and respond suboptimally to therapy. Currently, appropriate model systems to study the aggressive nature of these tumors are lacking. The objective of this study was to establish a mouse xenograft model of endometrial tumors derived from patients in order to study the biological aggressive characteristics that underlie invasion and metastasis. Methods Endometrial tumor tissue fragments (1.5 mm×1.5 mm) from patients undergoing surgery, were transplanted under the renal capsule of NOD scid gamma mice. After 6–8 weeks, tumors were excised and serially transplanted into additional mice for propagation. Immunohistochemical analysis of the tumors was done for various tumor markers. Results Four cases of different subtypes of endometrial cancer were grown and propagated in mice. Three of the four tumor cases invaded into the kidneys and to adjacent organs. While all tumors exhibited minimal to no staining for estrogen receptor α, progesterone receptor staining was observed for tumor grafts. In addition, levels and localization of E-cadherin, cytokeratin and vimentin varied depending on subtype. Finally, all tumor xenografts stained positively for urokinase plasminogen activator while 3 tumor xenografts, which showed invasive characteristics, stained positively for urokinase plasminogen activator receptor. Conclusion Endometrial tumors transplanted under the renal capsule exhibit growth, invasion and local spread. These tumors can be propagated and used to study aggressive endometrial cancer.

The allosteric AKT inhibitor, MK2206, decreases tumor growth and invasion in patient derived xenografts of endometrial cancer

ABSTRACT The purpose of this study was to test the effect of MK2206, an allosteric inhibitor of AKT, on the growth and invasion of patient-derived xenografts (PDX) of endometrial cancer. Three PDX lines, USC1 (uterine serous), EEC2 (endometrioid grade 2) and EEC4 (endometrioid grade 3) of endometrial cancer were grafted under the renal capsule of NSG mice. After 2 weeks of tumor growth the mice were treated with vehicle or 120mg/kg MK2206 twice a week for 3 weeks. Growth of all 3 PDX lines of different type and grade was significantly inhibited in response to MK2206 compared with vehicle control. Histological analysis revealed invasion and spread of EEC2 and EEC4 tumors were significantly decreased with MK2206 treatment. Immunohistochemical analysis showed a decrease in Ki67 in EEC2 upon MK2206 treatment, while USC1 and EEC4 tumors did not show differences in Ki67 levels. PR levels were evident in EEC2 which dramatically increased upon MK2206 treatment. In vitro analysis of EEC4 and AN3CA cells showed a dose-dependent decrease in p(Ser473)-AKT and p(Thr308)-AKT with MK2206. Invasion of EEC4 and AN3CA cells also significantly decreased after 36h and 72h of MK2206 treatment. PDX tumors provide an appropriate model for the testing of compounds that incorporates the heterogeneous nature of endometrial cancer. Further studies to determine efficacy of MK2206 alone or in combination with other compounds can also identify predictors of response to these pathway inhibitors.

Modeling African American prostate adenocarcinoma by inducing defined genetic alterations in organoids

Genomic studies are rapidly identifying genetic alterations in human cancer, but functional validation of such alterations has been slow. Here, using human prostate cancer as a model, we have assessed the feasibility of engineering defined genetic alterations in well-known cancer driver genes to transform benign prostate epithelial organoids derived from African American men. Benign human prostate organoids were transduced with lentiviruses expressing MYC, shPTEN, shTP53 and AR, alone and in various combinations, to recapitulate prostate cancer development. Organoids expressing MYC, shPTEN, shTP53 and AR (denoted MPPA); MYC, shPTEN and shTP53 (MPP); or MYC (M) were significantly larger, had higher proliferation rates and demonstrated pathologically transformed morphology compared to organoids transduced with control lentivirus. Alterations in MYC, PTEN and TP53 also affected the rate of organoid basal-to-luminal differentiation in vitro. MPPA and MPP organoids expressed the clinical prostate cancer marker AMACR and developed prostate adenocarcinoma when grafted under the renal capsule in mice. These data indicate that genetic alterations commonly observed in human prostate cancer can be rapidly modeled in human organoid culture. Prostate cancer organoids provide a useful pre-clinical model for the evaluation of new candidate cancer genes, cancer disparities, and potentially for testing of novel therapeutic agents.Genomic studies are rapidly identifying genetic alterations in human cancer, but functional validation of such alterations has been slow. Here, using human prostate cancer as a model, we have assessed the feasibility of engineering defined genetic alterations in well-known cancer driver genes to transform benign prostate epithelial organoids derived from African American men. Benign human prostate organoids were transduced with lentiviruses expressing MYC, shPTEN, shTP53 and AR, alone and in various combinations, to recapitulate prostate cancer development. Organoids expressing MYC, shPTEN, shTP53 and AR (denoted MPPA); MYC, shPTEN and shTP53 (MPP); or MYC (M) were significantly larger, had higher proliferation rates and demonstrated pathologically transformed morphology compared to organoids transduced with control lentivirus. Alterations in MYC, PTEN and TP53 also affected the rate of organoid basal-to-luminal differentiation in vitro. MPPA and MPP organoids expressed the clinical prostate cancer marker AMACR and developed prostate adenocarcinoma when grafted under the renal capsule in mice. These data indicate that genetic alterations commonly observed in human prostate cancer can be rapidly modeled in human organoid culture. Prostate cancer organoids provide a useful pre-clinical model for the evaluation of new candidate cancer genes, cancer disparities, and potentially for testing of novel therapeutic agents.

Anaplastic Lymphoma Kinase Mutation (ALK F1174C) in Small Cell Carcinoma of the Prostate and Molecular Response to Alectinib

Purpose: Small cell carcinoma of the prostate (SCCP) is an aggressive disease that can arise de novo or by transdifferentiation from prostate adenocarcinoma. Alterations in anaplastic lymphoma kinase (ALK) gene are involved in neuroblastoma, lung cancer, and other malignancies, but its role in SCCP has not been documented. We describe a patient with refractory de novo SCCP with ALK F1174C–activating mutation who obtained clinical benefit from treatment with ALK inhibitor. Experimental Design: Next-generation sequencing (NGS) was used to analyze primary and circulating tumor DNA (ctDNA). Prostate cancer databases were queried for alterations in ALK gene, mRNA, and its impact in clinical outcomes. In vitro prostate cell line/organoid models were generated by lentiviral-mediated expression of ALK and ALK F1174C and assessed for response to ALK inhibitors crizotinib and alectinib. Results: NGS analysis of the primary tumor and ctDNA of a 39-year-old patient with refractory SSCP identified ALK F1174C mutation. Treatment with second-generation ALK inhibitor alectinib resulted in radiographic stable disease for over 6 months, symptomatic improvement, and significant molecular response as reflected by declining ctDNA allele fraction. Analysis of prostate cancer datasets showed that ALK amplification was associated with poor outcome. In prostate cancer cells and organoids, ALK F1174C expression enhanced growth and induced expression of the neuroendocrine marker neuron-specific enolase. Alectinib was more effective than crizotinib in inhibiting ALK F1174C–expressing cell growth. Conclusions: These findings implicate ALK-activating mutations in SCCP pathogenesis and suggest the therapeutic potential of targeting ALK molecular alterations in some patients with SCCP. Clin Cancer Res; 24(12); 2732–9. ©2018 AACR.