These data indicate that progesterone exerts differential actions about phosphorylation of signaling pathway components and the growth in TSC2-deficient cells in nutrient-poor conditions. To determine whether patient-derived cells require steroids to grow in nutrient-rich conditions, we cultured these cells in complete media (containing steroids) or in phenol red-free media supplemented with charcoal-dextran stripped FBS (depleting steroids) for 72 hr, and measured cell growth at 24, 48 and 72 hr post cell seeding. growth of xenograft tumors; however, progesterone treatment did not affect the development of xenograft tumors of Tsc2-deficient cells. Importantly, treatment of progesterone plus estradiol resulted in alteration of lung morphology, and significantly improved the number of lung micrometastases of Tsc2-deficient cells compared with estradiol treatment only. Collectively, these data indicate that progesterone increases the metastatic potential of TSC2-deficient LAM patient-derived cells in vitro and lung metastasis in vivo. Thus, focusing on progesterone-mediated signaling events may have restorative benefit for LAM and possibly additional hormonally dependent cancers. ARS-853 or heterozygous mice [26]. Furthermore, inside a recently developed uterine-specific knockout mouse model, estradiol treatment improved myometrial proliferation, which was suppressed by ovariectomy and aromatase inhibition. Interestingly, progesterone treatment did not impact the proliferation of myometrial [24]. Despite these findings, the effect of progesterone within the proliferation, survival, and metastasis of cells lacking TSC2 has not been extensively investigated. We report here that progesterone treatment or progesterone plus estradiol activated Akt and ERK1/2 signaling pathways in LAM patient-derived cells. Importantly, progesterone only or in combination with ARS-853 estradiol strongly enhanced the migration and invasiveness of TSC2-deficient cells. In addition, treatment of progesterone plus estradiol synergistically decreased the cellular levels of reactive oxygen varieties (ROS), and enhanced cell survival under oxidative stress. Furthermore, treatment of progesterone plus estradiol advertised the growth of xenograft tumors; however, progesterone treatment did not affect the development of xenograft tumors of Tsc2-deficient ARS-853 cells. Importantly, treatment of progesterone plus estradiol advertised the lung metastasis of Tsc2-deficient cells compared with estradiol treatment only. Collectively, these data demonstrate that progesterone, in addition to estradiol, increases the metastatic potential of TSC2-deficient LAM patient-derived cells in vitro and lung metastasis in vivo. Therefore, focusing on progesterone-mediated signaling and/or cellular events may have restorative benefit for LAM and possibly additional hormonally dependent neoplasm. Results Progesterone activates ERK1/2 ARS-853 and Akt and enhances the proliferation of TSC2-deficient cells LAM patient-associated angiomyolipoma-derived cells and rat uterine leiomyoma-derived cells communicate estrogen receptor alpha (ER) and progesterone receptor (PgR), and respond to estradiol activation [27, 28]. The patient-derived cells ARS-853 were developed from a sporadic LAM-associated renal angiomyolipoma. These cells carry bi-allelic mutations of the TSC2 gene that are identical to the mutations found in the individuals pulmonary LAM cells [28]. The rat cells were developed from an Eker rat uterine leiomyoma, which is composed of smooth muscle mass cells lacking practical TSC2 [27, 29]. To validate the manifestation of ER and PgR, we measured their transcript levels using quantitative RT-PCR. The relative transcript Rabbit Polyclonal to TRXR2 level of ER was 4-fold higher in 621-101 cells (CT = 32.5) relative to normal human lung bronchial epithelial cells (BEAS-2B) (CT = 31.6) (Number 1A). Interestingly, the transcript level of ER was much lower in 621-101 cells relative to that in breast tumor MCF-7 cells (CT = 24.5) (Figure 1A). Moreover, the transcript level of PgR was detectable in 621-101 cells (CT = 31.6), although the value was lower than that of MCF-7 cells (CT = 22.2) (Number 1A). Furthermore, the manifestation of ER (CT = 34.5) and PgR (CT = 23.8) was confirmed in rat uterine leiomyoma-derived ELT3 cells (Number 1A), consistent with previous findings [27, 28]. To further determine the build up of ER.