generation-of-multicellular-human-primary-endometrial-organoids

Generation of Multicellular Human Primary Endometrial Organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...

Northwestern University

The forkhead box O1A (FOXO1A) has been identified as one gene that is up-regulated early in the decidualization process. To further investigate the role of FOXO1A during this process, six genes, IGFBP1, PRL, TIMP3, LAMB1, CNR1, and DCN, shown to be up-regulated during decidualization, were chosen as potential targets of FOXO1A action. Treatment of human endometrial stromal cells with hormones (estradiol and medroxyprogesterone acetate) plus dibutyryl cAMP (H+dbcAMP) for 48 h increased expression of IGFBP1, PRL, TIMP3, CNR1, and DCN but not LAMB1, as measured by real-time PCR. Silencing of FOXO1A using small interfering RNA oligonucleotides decreased IGFBP1 and DCN levels and increased CNR1, TIMP3, and PRL levels. LAMB1 was not affected. When FOXO1A was overexpressed in human endometrial stromal cells, expression of IGFBP1, DCN, and PRL increased, whereas levels of TIMP3 and CNR1 decreased. Addition of H+dbcAMP caused an increased expression of IGFBP1, PRL, and DCN beyond that of FOXO1A alone. TIMP3 and CNR1 levels decreased even further in response to H+dbcAMP compared with FOXO1A alone. LAMB1, which was unresponsive to FOXO1A, decreased when H+dbcAMP was added. Overexpressing FOXO1A also caused a change in cell shape, in that the stromal fibroblasts acquired a rounded, epithelioid appearance. Finally, reporter studies showed that cotransfection of FOXO1A significantly increased PRL promoter activity but not TIMP3 promoter activity. Addition of H+dbcAMP resulted in a significant increase in PRL promoter activity and a significant decrease in TIMP3 promoter activity. In summary, this study demonstrates the versatile nature of FOXO1A in the regulation of a number of decidualization-specific genes.

FOXO1A differentially regulates genes of decidualization.

Progesterone has been associated with promoting growth of uterine leiomyomas. The mechanisms involved remain unclear.

Progestins activate the AKT pathway in leiomyoma cells and promote survival.

Progesterone is a growth inhibitory hormone in the endometrium. While progestins can be used for the treatment of well-differentiated endometrial cancers, resistance to progestin therapy occurs for reasons that remain unclear. We have previously demonstrated that progesterone receptors (PR) A and B differentially regulate apoptosis in response to overexpression of the forkhead transcription factor, FOXO1. In this study, we further examined the PR-isoform-dependent cellular response to the AKT pathway. Treatment of PRA and PRB-expressing Ishikawa cells (PRA14, PRB23), with an AKT inhibitor API-59CJ-OMe (API-59) promoted apoptosis in the presence and absence of the ligand, R5020 preferentially in PRA14 cells. Upon PR knockdown using small interfering RNA, an increase in apoptosis was observed in PRB23 cells treated with API-59 with or without R5020 while there was no influence in PRA14 cells. Using an apoptosis-focused real-time PCR array, genes regulated by API-59 and R5020 were identified both common and unique to PRA14 and PRB23 cells. BIRC3 was identified as the only gene regulated by R5020 which occurred only in PRB cells. Knockdown of BIRC3 in PRB23 cells promoted a decrease in cell viability in response to API-59 + R5020. Furthermore, the important role of inhibitors of apoptosis (IAPs) in the PRB23 cells to promote cell survival was demonstrated using an antagonist to IAPs, a second mitochondria-derived activator of caspase (Smac also known as DIABLO) mimetic. Treatment of PRB23 cells with Smac mimetic increased apoptosis in response to API-59 + R5020. In summary, our findings indicate a mechanism by which PRB can promote cell survival in the setting of high AKT activity in endometrial cancer cells.

Progesterone receptor-B induction of BIRC3 protects endometrial cancer cells from AP1-59-mediated apoptosis.

Endometriosis affects approximately 10% of women in the United States and causes pain and infertility. Decidualization of endometrial stromal cells from women with endometriosis is aberrant.

Increased activation of the PI3K/AKT pathway compromises decidualization of stromal cells from endometriosis.

Progestin resistance is a major obstacle to treating early stage, well-differentiated endometrial cancer as well as recurrent endometrial cancer. The mechanism behind the suboptimal response to progestin is not well understood. The PTEN tumor suppressor gene is frequently mutated in type I endometrial cancers and this mutation results in hyperactivation of the PI3K/AKT pathway. We hypothesized that increased activation of AKT promotes an inadequate response to progestins in endometrial cancer cells. Ishikawa cells stably transfected with progesterone receptor B (PRB23 cells) were treated with the AKT inhibitor, MK-2206, which effectively decreased levels of p(Ser473)-AKT in a dose-dependent (10 nM to 1 uM) and time-dependent manner (0.5 h to 24 h). MK-2206 inhibited levels of p(Thr308)-AKT and a downstream target, p(Thr246)-PRAS40, but did not change levels of p(Thr202/Tyr204)ERK or p(Thr13/Tyr185)SAPK/JNK, demonstrating specificity of MK-2206 for AKT. Additionally, MK-2206 treatment of PRB23 cells resulted in a significant increase in levels of progesterone receptor B (PRB) protein. Microarray analysis of PRB23 cells identified PDK4 as the most highly upregulated gene among 70 upregulated genes in response to R5020. Inhibition of AKT further upregulated progestin-mediated expression of PDK4 but did not affect another progestin-responsive gene, SGK1. Treatment of PRB23 cells with R5020 and MK-2206 independently decreased viability of cells while the combination of R5020 and MK-2206 caused the greatest decrease in cell viability. Furthermore, mice with xenografted tumors treated with MK-2206 alone or with progesterone alone exhibited modest reductions in their tumor volume. The largest decrease in tumor size was observed in the mice treated with both MK-2206 and progesterone; these tumors exhibited the least proliferation (Ki67) and the most apoptosis (cleaved caspase-3) of all the treatment groups. In summary, inhibition of AKT stabilizes the Progesterone Receptor B and augments progesterone response in endometrial cancer cells that have hyperactivated AKT.

Inhibition of AKT with the orally active allosteric AKT inhibitor, MK-2206, sensitizes endometrial cancer cells to progestin.

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.

Increased AKT or MEK1/2 activity influences progesterone receptor levels and localization in endometriosis.

Uterine leiomyomas (fibroids) are a major public health problem. Current medical treatments with GnRH analogs do not provide long-term benefit. Thus, permanent shrinkage or inhibition of fibroid growth via medical means remains a challenge. The AKT pathway is a major growth and survival pathway for fibroids. We propose that AKT inhibition results in a transient regulation of specific mechanisms that ultimately drive cells into cellular senescence or cell death. In this study, we investigated specific mechanisms of AKT inhibition that resulted in senescence. We observed that administration of MK-2206, an allosteric AKT inhibitor, increased levels of reactive oxygen species, up-regulated the microRNA miR-182 and several senescence-associated genes (including p16, p53, p21, and β-galactosidase), and drove leiomyoma cells into stress-induced premature senescence (SIPS). Moreover, induction of SIPS was mediated by HMGA2, which colocalized to senescence-associated heterochromatin foci. This study provides a conceivable molecular mechanism of SIPS by AKT inhibition in fibroids. 

Article	Year
FOXO1A differentially regulates genes of decidualization. Endocrinology\| PubMed ID: 16690806	2006
Progestins activate the AKT pathway in leiomyoma cells and promote survival. The Journal of clinical endocrinology and metabolism\| PubMed ID: 19240153	2009
Progesterone receptor-B induction of BIRC3 protects endometrial cancer cells from AP1-59-mediated apoptosis. Hormones & cancer\| PubMed ID: 21760855	2011
Increased activation of the PI3K/AKT pathway compromises decidualization of stromal cells from endometriosis. The Journal of clinical endocrinology and metabolism\| PubMed ID: 22072736	2012
Inhibition of AKT with the orally active allosteric AKT inhibitor, MK-2206, sensitizes endometrial cancer cells to progestin. PloS one\| PubMed ID: 22911820	2012
Increased AKT or MEK1/2 activity influences progesterone receptor levels and localization in endometriosis. The Journal of clinical endocrinology and metabolism\| PubMed ID: 24064688	2013
Inactivation of AKT induces cellular senescence in uterine leiomyoma. Endocrinology\| PubMed ID: 24476133	2014

Zhenxiao Lu

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