BRCA1 mutation influences progesterone response in human benign mammary organoids.

BACKGROUND: Women, who carry a germline BRCA1 gene mutation, have a markedly increased risk of developing breast cancer during their lifetime. While BRCA1 carriers frequently develop triple-negative, basal-like, aggressive breast tumors, hormone signaling is important in the genesis of BRCA1 mutant breast cancers. We investigated the hormone response in BRCA1-mutated benign breast tissue using an in vitro organoid system. METHODS: Scaffold-free, multicellular human breast organoids generated from benign breast tissues from non-carrier or BRCA1 mutation carriers were treated in vitro with a stepwise menstrual cycle hormone regimen of estradiol (E2) and progesterone (P4) over the course of 28 days. RESULTS: Breast organoids exhibited characteristics of the native breast tissue, including expression of hormone receptors, collagen production, and markers of luminal and basal epithelium, and stromal fibroblasts. RNA sequencing analysis revealed distinct gene expression in response to hormone treatment in the non-carrier and BRCA1-mutated organoids. The selective progesterone receptor modulator, telapristone acetate (TPA), was used to identify specifically PR regulated genes. Specifically, extracellular matrix organization genes were regulated by E2+P4+TPA in the BRCA1-mutated organoids but not in the non-carrier organoids. In contrast, in the non-carrier organoids, known PR target genes such as the cell cycle genes were inhibited by TPA. CONCLUSIONS: These data show that BRCA1 mutation influences hormone response and in particular PR activity which differs from that of non-carrier organoids. Our organoid model system revealed important insights into the role of PR in BRCA1-mutated benign breast cells and the critical paracrine actions that modify hormone receptor (HR)-negative cells. Further analysis of the molecular mechanism of BRCA1 and PR crosstalk is warranted using this model system.

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 also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

Mechanism of Telapristone Acetate (CDB4124) on Progesterone Receptor Action in Breast Cancer Cells.

Progesterone is a steroid hormone that plays an important role in the breast. Progesterone exerts its action through binding to progesterone receptor (PR), a transcription factor. Deregulation of the progesterone signaling pathway is implicated in the formation, development, and progression of breast cancer. Next-generation selective progesterone receptor modulators (SPRMs) have potent antiprogestin activity and are selective for PR, reducing the off-target effects on other nuclear receptors. To date, there is limited information on how the newer generation of SPRMs, specifically telapristone acetate (TPA), affect PR function at the molecular level. In this study, T47D breast cancer cells were used to investigate the molecular mechanism by which TPA antagonizes PR action. Global profiling of the PR cistrome and interactome was done with chromatin immunoprecipitation sequencing (ChIP-seq) and rapid immunoprecipitation mass spectrometry. Validation studies were done on key genes and interactions. Our results demonstrate that treatment with the progestin (R5020) alone resulted in robust PR recruitment to the chromatin, and addition of TPA reduced PR recruitment globally. TPA significantly changed coregulator recruitment to PR compared with R5020. Upon conservative analysis, three proteins (TRPS1, LASP1, and AP1G1) were identified in the R5020+TPA-treated group. Silencing TRPS1 with small interfering RNA increased PR occupancy to the known PR regulatory regions and attenuated the inhibition of gene expression after TPA treatment. TRPS1 silencing alleviated the inhibition of proliferation by TPA. In conclusion, TPA decreases PR occupancy on chromatin and recruits coregulators such as TRPS1 to the PR complex, thereby regulating PR target gene expression and associated cellular responses.

The IGF-1R/AKT pathway has opposing effects on Nutlin-3a-induced apoptosis.

Nutlin-3a is a small molecule MDM2 antagonist and potent activator of wild-type p53. Nutlin-3a disrupts MDM2 binding to p53, thus increasing p53 levels and allowing p53 to inhibit proliferation or induce cell death. Factors that control sensitivity to Nutlin-3a-induced apoptosis are incompletely understood. In this study we isolated cisplatin-resistant clones from MHM cells, an MDM2-amplified and p53 wild-type osteosarcoma cell line. Cisplatin resistance in these clones resulted in part from heightened activation of the IGF-1R/AKT pathway. Interestingly, these cisplatin resistant clones showed hyper-sensitivity to Nutlin-3a induced apoptosis. Increased Nutlin-3a sensitivity was associated with reduced authophagy flux and a greater increase in p53 levels in response to Nutlin-3a treatment. IGF-1R and AKT inhibitors further increased apoptosis by Nutlin-3a in parental MHM cells and the cisplatin-resistant clones, confirming IGF-1R/AKT signaling promotes apoptosis resistance. However, IGF-1R and AKT inhibitors also reduced p53 accumulation in Nutlin-3a treated cells and increased autophagy flux, which we showed can promote apoptosis resistance. We conclude the IGF-1R/AKT pathway has opposing effects on Nutlin-3a-induced apoptosis. First, it can inhibit apoptosis, consistent with its well-established role as a survival-signaling pathway. Second, it can enhance Nutlin-3a induced apoptosis through a combination of maintaining p53 levels and inhibiting pro-survival autophagy.

Crosstalk between the IGF-1R/AKT/mTORC1 pathway and the tumor suppressors p53 and p27 determines cisplatin sensitivity and limits the effectiveness of an IGF-1R pathway inhibitor.

The insulin-like growth factor-1 receptor (IGF-1R) signaling pathway is aberrantly activated in multiple cancers and can promote proliferation and chemotherapy resistance. Multiple IGF-1R inhibitors have been developed as potential therapeutics. However, these inhibitors have failed to increase patient survival when given alone or in combination with chemotherapy agents. The reason(s) for the disappointing clinical effect of these inhibitors is not fully understood. Cisplatin (CP) activated the IGF-1R/AKT/mTORC1 pathway and stabilized p53 in osteosarcoma (OS) cells. p53 knockdown reduced IGF-1R/AKT/mTORC1 activation by CP, and IGF-1R inhibition reduced the accumulation of p53. These data demonstrate positive crosstalk between p53 and the IGF-1R/AKT/mTORC1 pathway in response to CP. Further studies showed the effect of IGF-1R inhibition on CP response is dependent on p53 status. In p53 wild-type cells treated with CP, IGF-1R inhibition increased p53s apoptotic function but reduced p53-dependent senescence, and had no effect on long term survival. In contrast, in p53-null/knockdown cells, IGF-1R inhibition reduced apoptosis in response to CP and increased long term survival. These effects were due to p27 since IGF-1R inhibition stabilized p27 in CP-treated cells, and p27 depletion restored apoptosis and reduced long term survival. Together, the results demonstrate 1) p53 expression determines the effect of IGF-1R inhibition on cancer cell CP response, and 2) crosstalk between the IGF-1R/AKT/mTORC1 pathway and p53 and p27 can reduce cancer cell responsiveness to chemotherapy and may ultimately limit the effectiveness of IGF-1R pathway inhibitors in the clinic.

p53-regulated autophagy is controlled by glycolysis and determines cell fate.

The tumor suppressor p53 regulates downstream targets that determine cell fate. Canonical p53 functions include inducing apoptosis, growth arrest, and senescence. Non-canonical p53 functions include its ability to promote or inhibit autophagy and its ability to regulate metabolism. The extent to which autophagy and/or metabolic regulation determines cell fate by p53 is unclear. To address this, we compared cells resistant or sensitive to apoptosis by the p53 activator Nutlin-3a. In resistant cells, glycolysis was maintained upon Nutlin-3a treatment, and activated p53 promoted prosurvival autophagy. In contrast, in apoptosis sensitive cells activated p53 increased superoxide levels and inhibited glycolysis through repression of glycolytic pathway genes. Glycolysis inhibition and increased superoxide inhibited autophagy by repressing ATG genes essential for autophagic vesicle maturation. Inhibiting glycolysis increased superoxide and blocked autophagy in apoptosis-resistant cells, causing p62-dependent caspase-8 activation. Finally, treatment with 2-DG or the autophagy inhibitors chloroquine or bafilomycin A1 sensitized resistant cells to Nutlin-3a-induced apoptosis. Together, these findings reveal novel links between glycolysis and autophagy that determine apoptosis-sensitivity in response to p53. Specifically, the findings indicate 1) that glycolysis plays an essential role in autophagy by limiting superoxide levels and maintaining expression of ATG genes required for autophagic vesicle maturation, 2) that p53 can promote or inhibit autophagy depending on the status of glycolysis, and 3) that inhibiting protective autophagy can expand the breadth of cells susceptible to Nutlin-3a induced apoptosis.

Isolation and characterization of an interactive culture of two Paenibacillus species with moderately thermophilic desulfurization ability.

OBJECTIVE: To isolate and characterize novel thermophilic bacteria capable of biodesulfurization of petroleum. RESULTS: A culture containing two Paenibacillus spp. (denoted "32O-W" and "32O-Y") was isolated by repeated passage of a soil sample at up to 55 °C in medium containing dibenzothiophene (DBT) as sulfur source. Only 32O-Y metabolized DBT, apparently via the 4S pathway; maximum activity occurred from 40 to 45 °C, with some activity up to at least 50 °C. 32O-W enhanced DBT metabolism by 32O-Y (by 22-74 % at 40-50 °C). With sulfate as sulfur source, 32O-Y and 32O-W grew well up to 58 and 63 °C, respectively. Selection of a mixed culture of 32O-Y and 32O-W at 54 °C increased DBT metabolism 36-42 % from 40 to 45 °C. Genome sequencing identified desulfurization gene homologs in the strains consistent with their desulfurization properties. CONCLUSION: The 32O-Y/32O-W culture may be a useful starting point for development of an improved thermophilic petroleum biodesulfurization process.

Novel roles for p53 in the genesis and targeting of tetraploid cancer cells.

Tetraploid (4N) cells are considered important in cancer because they can display increased tumorigenicity, resistance to conventional therapies, and are believed to be precursors to whole chromosome aneuploidy. It is therefore important to determine how tetraploid cancer cells arise, and how to target them. P53 is a tumor suppressor protein and key regulator of tetraploidy. As part of the "tetraploidy checkpoint", p53 inhibits tetraploid cell proliferation by promoting a G1-arrest in incipient tetraploid cells (referred to as a tetraploid G1 arrest). Nutlin-3a is a preclinical drug that stabilizes p53 by blocking the interaction between p53 and MDM2. In the current study, Nutlin-3a promoted a p53-dependent tetraploid G1 arrest in two diploid clones of the HCT116 colon cancer cell line. Both clones underwent endoreduplication after Nutlin removal, giving rise to stable tetraploid clones that showed increased resistance to ionizing radiation (IR) and cisplatin (CP)-induced apoptosis compared to their diploid precursors. These findings demonstrate that transient p53 activation by Nutlin can promote tetraploid cell formation from diploid precursors, and the resulting tetraploid cells are therapy (IR/CP) resistant. Importantly, the tetraploid clones selected after Nutlin treatment expressed approximately twice as much P53 and MDM2 mRNA as diploid precursors, expressed approximately twice as many p53-MDM2 protein complexes (by co-immunoprecipitation), and were more susceptible to p53-dependent apoptosis and growth arrest induced by Nutlin. Based on these findings, we propose that p53 plays novel roles in both the formation and targeting of tetraploid cells. Specifically, we propose that 1) transient p53 activation can promote a tetraploid-G1 arrest and, as a result, may inadvertently promote formation of therapy-resistant tetraploid cells, and 2) therapy-resistant tetraploid cells, by virtue of having higher P53 gene copy number and expressing twice as many p53-MDM2 complexes, are more sensitive to apoptosis and/or growth arrest by anti-cancer MDM2 antagonists (e.g. Nutlin).

Two 4N cell-cycle arrests contribute to cisplatin-resistance.

Cisplatin is a platinum-based drug that is used for the treatment of a wide-variety of primary human cancers. However, the therapeutic efficacy of cisplatin is often limited by intrinsic or acquired drug resistance. An important goal, therefore, is to identify mechanisms that lead to cisplatin resistance in cancer, and then use this information to more effectively target resistant cells. Cisplatin-resistant clones of the HCT116 cell line underwent a prolonged G2 arrest after cisplatin treatment while sensitive clones did not. The staurosporine analog UCN-01 abrogated this G2 arrest and sensitized the resistant clones to cisplatin. At later time points, 4N arrested cells assumed a tetraploid G1 state that was characterized by depletion of Cyclin A, Cyclin B, and CDC2, and increased expression of p53 and p21, in 4N cells. siRNA-mediated knockdown of p21 abrogated the tetraploid G1 arrest and induced killing that was dependent on p53. The results identify two targetable 4N arrests that can contribute to cisplatin resistance: First, a prolonged G2 arrest that can be targeted by UCN-01, and second, a tetraploid G1 arrest that can be targeted by siRNA against p21.