The crystal structure of a 70-kilodalton ribonucleoprotein complex from the central domain of the Thermus thermophilus 30S ribosomal subunit was solved at 2.6 angstrom resolution. The complex consists of a 104-nucleotide RNA fragment composed of two three-helix junctions that lie at the end of a central helix, and the ribosomal proteins S15, S6, and S18. S15 binds the ribosomal RNA early in the assembly of the 30S ribosomal subunit, stabilizing a conformational reorganization of the two three-helix junctions that creates the RNA fold necessary for subsequent binding of S6 and S18. The structure of the complex demonstrates the central role of S15-induced reorganization of central domain RNA for the subsequent steps of ribosome assembly.
Cell invasion is an initiating event during tumor cell metastasis and an essential process during development. A screen of C. elegans orthologs of genes over-expressed in invasive human melanoma cells has identified several components of the conserved DNA pre-replication complex (pre-RC) as positive regulators of anchor cell (AC) invasion. The pre-RC functions cell-autonomously in the G1-arrested AC to promote invasion, independently of its role in licensing DNA replication origins in proliferating cells. While the helicase activity of the pre-RC is necessary for AC invasion, the downstream acting DNA replication initiation factors are not required. The pre-RC promotes the invasive fate by regulating the expression of extracellular matrix genes and components of the PI3K signaling pathway. Increasing PI3K pathway activity partially suppressed the AC invasion defects caused by pre-RC depletion, suggesting that the PI3K pathway is one critical pre-RC target. We propose that the pre-RC acts in the non-proliferating AC as a transcriptional regulator that facilitates the switch to an invasive phenotype.
Functional modeling of breast epithelial hierarchy and stromal-epithelial cell interactions has been difficult due to inability to obtain sufficient stem-progenitor-mature epithelial cells and stromal cells. The recently developed epithelial reprogramming assay has partially overcome this limitation, allowing propagation of epithelial cells with stem, luminal progenitor and mature cell features. However, characterizing stromal cells using this assay is difficult because irradiated fibroblasts which can be difficult to distinguish from stromal cells are needed as feeder layer. A recent study demonstrated expansion of airway basal stem cells without a feeder layer through pharmacologic inhibition of TGFβ/BMP/SMAD signaling. We sought to develop this method for culture and expansion of cells from normal and cancerous breast samples. With appropriate modifications to growth media, we were able to obtain normal and stromal cells from breast biopsies of healthy women. The expanded cell population included CD10+/EpCAM- basal/myoepithelial cells, CD49f+/EpCAM+ luminal progenitor cells, CD49f-/EpCAM+ mature luminal cells, CD73+/EpCAM+/CD90- rare endogenous pluripotent somatic stem cells, CD73+/CD90+/EpCAM- mesenchymal stem cells, ALCAM (CD166)+/EpCAM+ cells, CD44+/CD24- cells, CD44+/CD24+ cells and ALDFLUOR+ stem/luminal progenitor cells. Epithelial cells were KRT14+, KRT19+ or both further documenting heterogeneity within epithelial cell population. We have extended this technique to grow breast epithelial cells from high-risk patients including BRCA1 mutant-carriers, tumor-adjacent normal and tumor cells from the same patient, pleural effusions and liver metastasis from breast cancer patients. Phenotypic characterization showed differences in the differentiation state of adjacent-normal and tumor cells. Tumor cells from pleural effusions showed remarkable phenotypic heterogeneity with a fraction of these cells expressing estrogen receptor. The assay described here, therefore, is versatile and provides resources to model epithelial-stromal interactions under normal and cancerous conditions as well as for genomics and screening of drugs to target metastasis on an individual level. Susan G. Komen for the Cure and Department of Defense supported this work. Citation Format: Nakshatri H, Ananappa M, Prasad MS, Kumar B, Liu Y, Storniolo AM, Miller KD, Bhat-Nakshatri P. Dual TGFβ/BMP inhibition allows in vitro expansion of multiple cell types from normal and cancerous breast [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-07-08.
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