Hedgehog signaling enables repair of ribosomal DNA double-strand breaks.

Ribosomal DNA (rDNA) consists of highly repeated sequences that are prone to incurring damage. Delays or failure of rDNA double-strand break (DSB) repair are deleterious, and can lead to rDNA transcriptional arrest, chromosomal translocations, genomic losses, and cell death. Here, we show that the zinc-finger transcription factor GLI1, a terminal effector of the Hedgehog (Hh) pathway, is required for the repair of rDNA DSBs. We found that GLI1 is activated in triple-negative breast cancer cells in response to ionizing radiation (IR) and localizes to rDNA sequences in response to both global DSBs generated by IR and site-specific DSBs in rDNA. Inhibiting GLI1 interferes with rDNA DSB repair and impacts RNA polymerase I activity and cell viability. Our findings tie Hh signaling to rDNA repair and this heretofore unknown function may be critically important in proliferating cancer cells.

Novel role of the dietary flavonoid fisetin in suppressing rRNA biogenesis.

The nucleolus of a cell is a critical cellular compartment that is responsible for ribosome biogenesis and plays a central role in tumor progression. Fisetin, a nutraceutical, is a naturally occurring flavonol from the flavonoid group of polyphenols that has anti-cancer effects. Fisetin negatively impacts several signaling pathways that support tumor progression. However, effect of fisetin on the nucleolus and its functions were unknown. We observed that fisetin is able to physically enter the nucleolus. In the nucleolus, RNA polymerase I (RNA Pol I) mediates the biogenesis of ribosomal RNA. Thus, we investigated the impacts of fisetin on the nucleolus. We observed that breast tumor cells treated with fisetin show a 20-30% decreased nucleolar abundance per cell and a 30-60% downregulation of RNA Pol I transcription activity, as well as a 50-70% reduction in nascent rRNA synthesis, depending on the cell line. Our studies show that fisetin negatively influences MAPK/ERK pathway to impair RNA Pol I activity and rRNA biogenesis. Functionally, we demonstrate that fisetin acts synergistically (CI = 0.4) with RNA Pol I inhibitor, BMH-21 and shows a noteworthy negative impact (60% decrease) on lung colonization of breast cancer cells. Overall, our findings highlight the potential of ribosomal RNA (rRNA) biogenesis as a target for secondary prevention and possible treatment of metastatic disease.

The nucleolus: a central response hub for the stressors that drive cancer progression.

The nucleolus is a sub-nuclear body known primarily for its role in ribosome biogenesis. Increased number and/or size of nucleoli have historically been used by pathologists as a prognostic indicator of cancerous lesions. This increase in nucleolar number and/or size is classically attributed to the increased need for protein synthesis in cancer cells. However, evidences suggest that the nucleolus plays critical roles in many cellular functions in both normal cell biology and disease pathologies, including cancer. As new functions of the nucleolus are elucidated, there is mounting evidence to support the role of the nucleolus in regulating additional cellular functions, particularly response to cellular stressors, maintenance of genome stability, and DNA damage repair, as well as the regulation of gene expression and biogenesis of several ribonucleoproteins. This review highlights the central role of the nucleolus in carcinogenesis and cancer progression and discusses how cancer cells may become "addicted" to nucleolar functions.

Isolation of Nucleoli for Characterization of Nucleolar Contents to Uncover Clues to Metastatic Progression.

Nucleolar isolation is a crucial technique for the study of nucleolar contents and regulation of ribosome biogenesis. Lysed cells are spun through various concentrations of sucrose and magnesium chloride to separate the notoriously dense nucleoli from the rest of the cell. Here we describe isolation of nucleoli from the breast cancer cell line MDA-MB-468. The resulting nucleolar fraction is subjected to immunoblotting to confirm the purity of the nucleolar fraction.

Inhibiting ß-catenin disables nucleolar functions in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) patients with upregulated Wnt/ß-catenin signaling often have poor clinical prognoses. During pathological examinations of breast cancer sections stained for ß-catenin, we made the serendipitous observation that relative to non-TNBC, specimens from TNBC patients have a greater abundance of nucleoli. There was a remarkable direct relationship between nuclear ß-catenin and greater numbers of nucleoli in TNBC tissues. These surprising observations spurred our investigations to decipher the differential functional relevance of the nucleolus in TNBC versus non-TNBC cells. Comparative nucleolar proteomics revealed that the majority of the nucleolar proteins in TNBC cells were potential targets of ß-catenin signaling. Next, we undertook an analysis of the nucleolar proteome in TNBC cells in response to ß-catenin inhibition. This effort revealed that a vital component of pre-rRNA processing, LAS1 like ribosome biogenesis factor (LAS1L) was significantly decreased in the nucleoli of ß-catenin inhibited TNBC cells. Here we demonstrate that LAS1L protein expression is significantly elevated in TNBC patients, and it functionally is important for mammary tumor growth in xenograft models and enables invasive attributes. Our observations highlight a novel function for ß-catenin in orchestrating nucleolar activity in TNBCs.

Conditional knockout of N-Myc and STAT interactor disrupts normal mammary development and enhances metastatic ability of mammary tumors.

The process of organ development requires a delicate balance between cellular plasticity and differentiation. This balance is disrupted in cancer initiation and progression. N-Myc and STAT interactor (NMI: human or Nmi: murine) has emerged as a relevant player in the etiology of breast cancer. However, a fundamental understanding of its relevance to normal mammary biology is lacking. To gain insight into its normal function in mammary gland, we generated a mammary-specific Nmi knockout mouse model. We observed that Nmi protein expression is induced in mammary epithelium at the onset of pregnancy, in luminal cells and persists throughout lactation. Nmi knockout results in a precocious alveolar phenotype. These alveoli exhibit an extensive presence of nuclear ß-catenin and enhanced Wnt/ß-catenin signaling. The Nmi knockout pubertal ductal tree shows enhanced invasion of the mammary fatpad and increased terminal end bud numbers. Tumors from Nmi null mammary epithelium show a significant enrichment of poorly differentiated cells with elevated stem/progenitor markers, active Wnt/ß-catenin signaling, highly invasive morphology as well as, increased number of distant metastases. Our study demonstrates that Nmi has a distinct role in the differentiation process of mammary luminal epithelial cell compartment and developmental aberrations resulting from Nmi absence contribute to metastasis and demonstrates that aberration in normal developmental program can lead to metastatic disease, highlighting the contribution and importance of luminal progenitor cells in driving metastatic disease.

Use of an impedance threshold device in spontaneously breathing patients with hypotension secondary to trauma: an observational cohort feasibility study.

BACKGROUND: An impedance threshold device (ITD) intended for use in the spontaneously breathing patient has been shown to raise blood pressure in hypotensive patients. This device has not been evaluated in patients with hypotension secondary to trauma. This study focused on changes in key vital signs when the ITD was added to the paramedic treatment protocol for hypotensive patients with prehospital traumatic injury. METHODS: A 6-month prospective nonrandomized observational cohort study was conducted of 200 spontaneously breathing symptomatic adult patients with prehospital hypotension due to multiple causes; the patients of primary interest experienced a traumatic injury. Upon determination of hypotension (systolic blood pressure of approximately ≤90 mm Hg), standard therapy was initiated by application of the mask-style ITD. Vital signs were documented every 2 minutes to 5 minutes after intervention. A change in mean arterial pressure (MAP) with ITD use was the primary study endpoint. RESULTS: Of the 200 hypotensive subjects treated, 29 (3 were excluded because of incomplete data sets and 3 patients treated with the ITD were excluded because their blood pressure did not meet inclusion criterion) were hypotensive secondary to trauma. Their MAP increased from 60 mm Hg (SD, 11 mm Hg; 95% confidence interval [CI], 8.17-15.432) to 78 mm Hg (16 mm Hg; 95% CI, 12.43-23.46) (p = 0.001), without significant change in mean heart rate. Approximately 75% of the patients reported moderate to easy tolerance. Similar increases in MAP were observed in the nontraumatic patients, from 60 mm Hg (10 mm Hg; 95% CI, 9.4-11.5) to 70 (15; 95% CI, 13.4-16.7) (p = 0.0001). CONCLUSION: In this observational cohort study of patients with hypotension secondary to trauma, the ITD was well tolerated, and MAP as well as systolic and diastolic blood pressure were improved. The patients were not overresuscitated with this intervention. On the basis of these findings, additional studies in patients with hypotension secondary to traumatic injury should be performed to better define the need and benefit of additional fluid resuscitation when the ITD is used. LEVEL OF EVIDENCE: Therapeutic study, level IV.