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Browsing by Author "Padua, Maria B."
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Item Dependence receptor UNC5A restricts luminal to basal breast cancer plasticity and metastasis(BMC, 2018-05-02) Padua, Maria B.; Bhat-Nakshatri, Poornima; Anjanappa, Manjushree; Prasad, Mayuri S.; Hao, Yangyang; Rao, Xi; Liu, Sheng; Wan, Jun; Liu, Yunlong; McElyea, Kyle; Jacobsen, Max; Sandusky, George; Althouse, Sandra; Perkins, Susan; Nakshatri, Harikrishna; Surgery, School of MedicineBACKGROUND: The majority of estrogen receptor-positive (ERα+) breast cancers respond to endocrine therapies. However, resistance to endocrine therapies is common in 30% of cases, which may be due to altered ERα signaling and/or enhanced plasticity of cancer cells leading to breast cancer subtype conversion. The mechanisms leading to enhanced plasticity of ERα-positive cancer cells are unknown. METHODS: We used short hairpin (sh)RNA and/or the CRISPR/Cas9 system to knockdown the expression of the dependence receptor UNC5A in ERα+ MCF7 and T-47D cell lines. RNA-seq, quantitative reverse transcription polymerase chain reaction, chromatin immunoprecipitation, and Western blotting were used to measure the effect of UNC5A knockdown on basal and estradiol (E2)-regulated gene expression. Mammosphere assay, flow cytometry, and immunofluorescence were used to determine the role of UNC5A in restricting plasticity. Xenograft models were used to measure the effect of UNC5A knockdown on tumor growth and metastasis. Tissue microarray and immunohistochemistry were utilized to determine the prognostic value of UNC5A in breast cancer. Log-rank test, one-way, and two-way analysis of variance (ANOVA) were used for statistical analyses. RESULTS: Knockdown of the E2-inducible UNC5A resulted in altered basal gene expression affecting plasma membrane integrity and ERα signaling, as evident from ligand-independent activity of ERα, altered turnover of phosphorylated ERα, unique E2-dependent expression of genes effecting histone demethylase activity, enhanced upregulation of E2-inducible genes such as BCL2, and E2-independent tumorigenesis accompanied by multiorgan metastases. UNC5A depletion led to the appearance of a luminal/basal hybrid phenotype supported by elevated expression of basal/stem cell-enriched ∆Np63, CD44, CD49f, epidermal growth factor receptor (EGFR), and the lymphatic vessel permeability factor NTN4, but lower expression of luminal/alveolar differentiation-associated ELF5 while maintaining functional ERα. In addition, UNC5A-depleted cells acquired bipotent luminal progenitor characteristics based on KRT14+/KRT19+ and CD49f+/EpCAM+ phenotype. Consistent with in vitro results, UNC5A expression negatively correlated with EGFR expression in breast tumors, and lower expression of UNC5A, particularly in ERα+/PR+/HER2- tumors, was associated with poor outcome. CONCLUSION: These studies reveal an unexpected role of the axon guidance receptor UNC5A in fine-tuning ERα and EGFR signaling and the luminal progenitor status of hormone-sensitive breast cancers. Furthermore, UNC5A knockdown cells provide an ideal model system to investigate metastasis of ERα+ breast cancers.Item Pharmacological Dual Inhibition of Tumor and Tumor-Induced Functional Limitations in a Transgenic Model of Breast Cancer(American Association for Cancer Research, 2017-12) Wang, Ruizhong; Bhat-Nakshatri, Poornima; Padua, Maria B.; Prasad, Mayuri S.; Anjanappa, Manjushree; Jacobson, Max; Finnearty, Courtney; Sefcsik, Victoria; McElyea, Kyle; Redmond, Rachael; Sandusky, George; Penthala, Narsimha; Crooks, Peter A.; Liu, Jianguo; Zimmers, Teresa A.; Nakshatri, Harikrishna; Surgery, School of MedicineBreast cancer progression is associated with systemic effects, including functional limitations and sarcopenia without the appearance of overt cachexia. Autocrine/paracrine actions of cytokines/chemokines produced by cancer cells mediate cancer progression and functional limitations. The cytokine-inducible transcription factor NF-κB could be central to this process, as it displays oncogenic functions and is integral to the Pax7:MyoD:Pgc-1β:miR-486 myogenesis axis. We tested this possibility using the MMTV-PyMT transgenic mammary tumor model and the NF-κB inhibitor dimethylaminoparthenolide (DMAPT). We observed deteriorating physical and functional conditions in PyMT+ mice with disease progression. Compared with wild-type mice, tumor-bearing PyMT+ mice showed decreased fat mass, impaired rotarod performance, and reduced grip strength as well as increased extracellular matrix (ECM) deposition in muscle. Contrary to acute cachexia models described in the literature, mammary tumor progression was associated with reduction in skeletal muscle stem/satellite-specific transcription factor Pax7. Additionally, we observed tumor-induced reduction in Pgc-1β in muscle, which controls mitochondrial biogenesis. DMAPT treatment starting at 6 to 8 weeks age prior to mammary tumor occurrence delayed mammary tumor onset and tumor growth rates without affecting metastasis. DMAPT overcame cancer-induced functional limitations and improved survival, which was accompanied with restoration of Pax7, Pgc-1β, and mitochondria levels and reduced ECM levels in skeletal muscles. In addition, DMAPT restored circulating levels of 6 out of 13 cancer-associated cytokines/chemokines changes to levels seen in healthy animals. These results reveal a pharmacological approach for overcoming cancer-induced functional limitations, and the above-noted cancer/drug-induced changes in muscle gene expression could be utilized as biomarkers of functional limitations.Item Single cell RNA analysis of the left-right organizer transcriptome reveals potential novel heterotaxy genes(Springer Nature, 2023-07-01) Bellchambers, Helen M.; Phatak, Amruta R.; Nenni, Mardi J.; Padua, Maria B.; Gao, Hongyu; Liu, Yunlong; Ware, Stephanie M.; Pediatrics, School of MedicineThe establishment of left-right patterning in mice occurs at a transient structure called the embryonic node or left-right organizer (LRO). Previous analysis of the LRO has proven challenging due to the small cell number and transient nature of this structure. Here, we seek to overcome these difficulties to define the transcriptome of the LRO. Specifically, we used single cell RNA sequencing of 0-1 somite embryos to identify LRO enriched genes which were compared to bulk RNA sequencing of LRO cells isolated by fluorescent activated cell sorting. Gene ontology analysis indicated an enrichment of genes associated with cilia and laterality terms. Furthermore, comparison to previously identified LRO genes identified 127 novel LRO genes, including Ttll3, Syne1 and Sparcl1, for which the expression patterns were validated using whole mount in situ hybridization. This list of novel LRO genes will be a useful resource for further studies on LRO morphogenesis, the establishment of laterality and the genetic causes of heterotaxy.Item The genetic landscape of cardiovascular left–right patterning defects(Current Opinion in Genetics & Development, 2022) Wells, John R.; Padua, Maria B.; Ware, Stephanie M.; Medical and Molecular Genetics, School of MedicineHeterotaxy is a disorder with complex congenital heart defects and diverse left-right (LR) patterning defects in other organ systems. Despite evidence suggesting a strong genetic component in heterotaxy, the majority of molecular causes remain unknown. Established genes often involve a ciliated, embryonic structure known as the left-right organizer (LRO). Herein, we focus on genetic discoveries in heterotaxy in the past two years. These include complex genetic architecture, novel mechanisms regulating cilia formation, and evidence for conservation of LR patterning between distant species. We feature new insights regarding established LR signaling pathways, bring attention to heterotaxy candidate genes in novel pathways, and provide an extensive overview of genes previously associated with laterality phenotypes in humans.