Browsing by Author "Jacobsen, Max"
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Item Author Correction: Upregulation of lipid metabolism genes in the breast prior to cancer diagnosis(Springer Nature, 2024-06-17) Marino, Natascia; German, Rana; Rao, Xi; Simpson, Ed; Liu, Sheng; Wan, Jun; Liu, Yunlong; Sandusky, George; Jacobsen, Max; Stovall, Miranda; Cao, Sha; Storniolo, Anna Maria V.; Medicine, School of MedicineCorrection to: npj Breast Cancer 10.1038/s41523-020-00191-8, published online 06 October 2020 In this article, the author name Miranda Stovall was incorrectly written as Miranda Stoval. The original article has been corrected.Item Bidirectional Regulatory Cross-Talk between Cell Context and Genomic Aberrations Shapes Breast Tumorigenesis(American Association for Cancer Research, 2021) Kumar, Brijesh; Bhat-Nakshatri, Poornima; Maguire, Calli; Jacobsen, Max; Temm, Constance J.; Sandusky, George; Nakshatri, Harikrishna; Surgery, School of MedicineBreast cancers are classified into five intrinsic subtypes and 10 integrative clusters based on gene expression patterns and genomic aberrations, respectively. Although the cell-of-origin, adaptive plasticity, and genomic aberrations shape dynamic transcriptomic landscape during cancer progression, how interplay between these three core elements governs obligatory steps for a productive cancer progression is unknown. Here, we used genetic ancestry-mapped immortalized breast epithelial cell lines generated from breast biopsies of healthy women that share gene expression profiles of luminal A, normal-like, and basal-like intrinsic subtypes of breast cancers and breast cancer relevant oncogenes to develop breast cancer progression model. Using flow cytometry, mammosphere growth, signaling pathway, DNA damage response, and in vivo tumorigenicity assays, we provide evidence that establishes cell context-dependent effects of oncogenes in conferring plasticity, self-renewal/differentiation, intratumor heterogeneity, and metastatic properties. In contrast, oncogenic aberrations, independent of cell context, shaped response to DNA damage-inducing agents. Collectively, this study reveals how the same set of genomic aberration can have distinct effects on tumor characteristics based on cell-of-origin of tumor and highlights the need to utilize multiple "normal" epithelial cell types to decipher oncogenic properties of a gene of interest. In addition, by creating multiple isogenic cell lines ranging from primary cells to metastatic variants, we provide resources to elucidate cell-intrinsic properties and cell-oncogene interactions at various stages of cancer progression. IMPLICATIONS: Our findings demonstrate that how an interplay between the normal cell type that encountered genomic aberrations and type of genomic aberration influences heterogeneity, self-renewal/differentiation, and tumor properties including propensity for metastasis.Item CD68 Macrophage Expression in Normal Breast Tissue and Cancer(2019-04-19) Gaines, Madelynn; Jacobsen, Max; Temm, Connie; Sandusky, GeorgeBreast cancer is a common disease and is the second leading cause of death in women. This type of cancer is usually hormonally driven by estrogen, progesterone, and HER2. Macrophages play a large role in the tumor microenvironment (TME). The aim of this study was to investigate the percent of macrophages in 32 normal breast tissues, 66 normal adjacent tissue (NAT), and 82 breast cancer tissues using the CD68-specific biomarker. Tissue microarrays (TMA) were created, which are composed of 2-mm cores from multiple patients mounted onto a single slide. The breast tissue samples were fixed, processed, microtomed, and stained with CD68. Unstained slides were immunostained using the Dako FLEX system. The slides were imaged using the Aperio Whole Slide Imaging platform and the tissues were evaluated using the positive pixel count algorithm (a quantitative image analysis system). The positivity of macrophages in the tissue samples were reported as a percentage, and compared across the three groups. It was found that the CD68 positivity in the normal and breast cancer tissue were even, and the NAT was lower. However, the three groups had overlapping standard deviations. Because difference between the percentages of each group was minimal and the deviations overlapped, it was concluded that there is no statistical difference between the three groups.Item Cryo-EM structures of prion protein filaments from Gerstmann-Sträussler-Scheinker disease(Springer, 2022) Hallinan, Grace I.; Ozcan, Kadir A.; Hoq, Md Rejaul; Cracco, Laura; Vago, Frank S.; Bharath, Sakshibeedu R.; Li, Daoyi; Jacobsen, Max; Doud, Emma H.; Mosley, Amber L.; Fernandez, Anllely; Garringer, Holly J.; Jiang, Wen; Ghetti, Bernardino; Vidal, Ruben; Pathology and Laboratory Medicine, School of MedicinePrion protein (PrP) aggregation and formation of PrP amyloid (APrP) are central events in the pathogenesis of prion diseases. In the dominantly inherited prion protein amyloidosis known as Gerstmann-Sträussler-Scheinker (GSS) disease, plaques made of PrP amyloid are present throughout the brain. The c.593t > c mutation in the prion protein gene (PRNP) results in a phenylalanine to serine amino acid substitution at PrP residue 198 (F198S) and causes the most severe amyloidosis among GSS variants. It has been shown that neurodegeneration in this disease is associated with the presence of extracellular APrP plaques and neuronal intracytoplasmic Tau inclusions, that have been shown to contain paired helical filaments identical to those found in Alzheimer disease. Using cryogenic electron microscopy (cryo-EM), we determined for the first time the structures of filaments of human APrP, isolated post-mortem from the brain of two symptomatic PRNP F198S mutation carriers. We report that in GSS (F198S) APrP filaments are composed of dimeric, trimeric and tetrameric left-handed protofilaments with their protomers sharing a common protein fold. The protomers in the cross-β spines consist of 62 amino acids and span from glycine 80 to phenylalanine 141, adopting a previously unseen spiral fold with a thicker outer layer and a thinner inner layer. Each protomer comprises nine short β-strands, with the β1 and β8 strands, as well as the β4 and β9 strands, forming a steric zipper. The data obtained by cryo-EM provide insights into the structural complexity of the PrP filament in a dominantly inherited human PrP amyloidosis. The novel findings highlight the urgency of extending our knowledge of the filaments' structures that may underlie distinct clinical and pathologic phenotypes of human neurodegenerative diseases.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 Genetic Ancestry–dependent Differences in Breast Cancer–induced Field Defects in the Tumor-adjacent Normal Breast(AACR, 2019-05) Nakshatri, Harikrishna; Kumar, Brijesh; Burney, Heather; Cox, Mary L.; Jacobsen, Max; Sandusky, George; D’Souza-Schorey, Crislyn; Storniolo, Anna Maria; Biostatistics, School of Public HealthPurpose: Genetic ancestry influences evolutionary pathways of cancers. However, whether ancestry influences cancer-induced field defects is unknown. The goal of this study was to utilize ancestry-mapped true normal breast tissues as controls to identify cancer-induced field defects in normal tissue adjacent to breast tumors (NATs) in women of African American (AA) and European (EA) ancestry. Experimental Design: A tissue microarray comprising breast tissues of ancestry-mapped 100 age-matched healthy women from the Komen Tissue Bank (KTB) at Indiana University (Indianapolis, IN) and tumor-NAT pairs from 100 women (300 samples total) was analyzed for the levels of ZEB1, an oncogenic transcription factor that is central to cell fate, mature luminal cell–enriched estrogen receptor alpha (ERα), GATA3, FOXA1, and for immune cell composition. Results: ZEB1+ cells, which were localized surrounding the ductal structures of the normal breast, were enriched in the KTB-normal of AA compared with KTB-normal of EA women. In contrast, in EA women, both NATs and tumors compared with KTB-normal contained higher levels of ZEB1+ cells. FOXA1 levels were lower in NATs compared with KTB-normal in AA but not in EA women. We also noted variations in the levels of GATA3, CD8+ T cells, PD1+ immune cells, and PDL1+ cell but not CD68+ macrophages in NATs of AA and EA women. ERα levels did not change in any of our analyses, pointing to the specificity of ancestry-dependent variations. Conclusions: Genetic ancestry–mapped tissues from healthy individuals are required for proper assessment and development of cancer-induced field defects as early cancer detection markers. This finding is significant in light of recent discoveries of influence of genetic ancestry on both normal biology and tumor evolution.Item Identification of LIMK2 as a therapeutic target in castration resistant prostate cancer(Elsevier, 2019-04) Nikhil, Kumar; Chang, Lei; Viccaro, Keith; Jacobsen, Max; McGuire, Callista; Satapathy, Shakti R.; Tandiary, Michael; Broman, Meaghan M.; Cresswell, Gregory; He, Yizhou J.; Sandusky, George E.; Ratliff, Timothy L.; Chowdhury, Dipanjan; Shah, Kavita; Pathology and Laboratory Medicine, School of MedicineThis study identified LIMK2 kinase as a disease-specific target in castration resistant prostate cancer (CRPC) pathogenesis, which is upregulated in response to androgen deprivation therapy, the current standard of treatment for prostate cancer. Surgical castration increases LIMK2 expression in mouse prostates due to increased hypoxia. Similarly, human clinical specimens showed highest LIMK2 levels in CRPC tissues compared to other stages, while minimal LIMK2 was observed in normal prostates. Most notably, inducible knockdown of LIMK2 fully reverses CRPC tumorigenesis in castrated mice, underscoring its potential as a clinical target for CRPC. We also identified TWIST1 as a direct substrate of LIMK2, which uncovered the molecular mechanism of LIMK2-induced malignancy. TWIST1 is strongly associated with CRPC initiation, progression and poor prognosis. LIMK2 increases TWIST1 mRNA levels upon hypoxia; and stabilizes TWIST1 by direct phosphorylation. TWIST1 also stabilizes LIMK2 by inhibiting its ubiquitylation. Phosphorylation-dead TWIST1 acts as dominant negative and fully prevents EMT and tumor formation in vivo, thereby highlighting the significance of LIMK2-TWIST1 signaling axis in CRPC. As LIMK2 null mice are viable, targeting LIMK2 should have minimal collateral toxicity, thereby improving the overall survival of CRPC patients.Item Immune Reconstitution and Thymic Involution in the Acute and Delayed Hematopoietic Radiation Syndromes(Wolters Kluwer, 2020) Wu, Tong; Plett, P. Artur; Chua, Hui Lin; Jacobsen, Max; Sandusky, George E.; MacVittie, Thomas J.; Orschell, Christie M.; Medicine, School of MedicineLymphoid lineage recovery and involution after exposure to potentially lethal doses of ionizing radiation have not been well defined, especially the long-term effects in aged survivors and with regard to male / female differences. To examine these questions, male and female C57BL/6 mice were exposed to lethal radiation at 12 weeks of age in a model of the Hematopoietic-Acute Radiation Syndrome, and bone marrow, thymus, spleen and peripheral blood examined up to 24 months of age for the lymphopoietic Delayed Effects of Acute Radiation Exposure. Aged mice showed myeloid skewing and incomplete lymphocyte recovery in all lymphoid tissues. Spleen and peripheral blood both exhibited a mono-phasic recovery pattern while thymus demonstrated a bi-phasic pattern. Naïve T cells in blood and spleen and all subsets of thymocytes were decreased in aged irradiated mice compared to age-matched non-irradiated controls. Of interest, irradiated males experienced significantly improved reconstitution of thymocyte subsets and peripheral blood elements compared to females. Bone marrow from aged irradiated survivors was significantly deficient in the primitive lymphoid-primed multipotent progenitors and common lymphoid progenitors, which were only 8–10% of levels in aged-matched non-irradiated controls. Taken together, these analyses define significant age- and sex-related deficiencies at all levels of lymphopoiesis throughout the lifespan of survivors of the Hematopoietic-Acute Radiation Syndrome, and may provide a murine model suitable for assessing the efficacy of potential medical countermeasures and therapeutic strategies to alleviate the severe immune suppression that occurs after radiation exposure.Item Ketotifen Modulates Mast Cell Chemotaxis to Kit-Ligand, but Does Not Impact Mast Cell Numbers, Degranulation, or Tumor Behavior in Neurofibromas of Nf1-Deficient Mice(American Association for Cancer Research, 2019-12-01) Burks, Ciersten A.; Rhodes, Steven D.; Bessler, Waylan K.; Chen, Shi; Smith, Abbi; Gehlhausen, Jeffrey R.; Hawley, Eric T.; Jiang, Li; Li, Xiaohong; Yuan, Jin; Lu, Qingbo; Jacobsen, Max; Sandusky, George E.; Jones, David R.; Clapp, D. Wade; Blakeley, Jaishri O.; Pediatrics, School of MedicineNeurofibromatosis Type 1 (NF1) is one of the most common genetic tumor predisposition syndromes in humans. Mutant NF1 results in dysregulated RAS allowing neoplasms throughout the neuroaxis. Plexiform neurofibromas (pNFs) afflict up to 50% of patients with NF1. They are complex tumors of the peripheral nerve that cause major morbidity via nerve dysregulation and mortality via conversion to malignant sarcoma. Genetically engineered mouse models (GEMMs) of NF1 provide valuable insights for the identification of therapies that have utility in people with pNF. Preclinical studies in GEMMs implicate mast cells and the c-Kit/Kit ligand pathway in pNF tumorigenesis. Kit ligand is a potent chemokine secreted by tumorigenic, Nf1-deficient Schwann cells. Ketotifen is an FDA-approved drug for the treatment of allergic conjunctivitis and asthma that promotes mast cell stabilization and has been used in prior case studies to treat or prevent pNFs. This study investigated the effect of ketotifen on mast cell infiltration and degranulation in the presence and absence of Kit ligand provocation and the effect of ketotifen on shrinking or preventing pNF formation in the Nf1flox/flox;PostnCre+ GEMM. Ketotifen decreased mast cell infiltration in response to exogenous Kit ligand administration, but did not affect mast cell degranulation. Importantly, ketotifen did not reduce mast cells numbers or activity in pNF and did not prevent pNF formation or decrease the volume of established pNF despite administration of pharmacologically active doses. These findings suggest ketotifen has limited use as monotherapy to prevent or reduce pNF burden in the setting of Nf1 mutations.Item Pharmacological inhibition of Carbonic Anhydrase IX and XII to enhance targeting of acute myeloid leukaemia cells under hypoxic conditions(Wiley, 2021-12) Chen, Fangli; Licarete, Emilia; Wu, Xue; Petrusca, Daniela; Maguire, Callista; Jacobsen, Max; Colter, Austyn; Sandusky, George E.; Czader, Magdalena; Capitano, Maegan L.; Ropa, James P.; Boswell, H. Scott; Carta, Fabrizio; Supuran, Claudiu T.; Parkin, Brian; Fishel, Melissa L.; Konig, Heiko; Pathology and Laboratory Medicine, School of MedicineAcute myeloid leukaemia (AML) is an aggressive form of blood cancer that carries a dismal prognosis. Several studies suggest that the poor outcome is due to a small fraction of leukaemic cells that elude treatment and survive in specialised, oxygen (O2 )-deprived niches of the bone marrow. Although several AML drug targets such as FLT3, IDH1/2 and CD33 have been established in recent years, survival rates remain unsatisfactory, which indicates that other, yet unrecognized, mechanisms influence the ability of AML cells to escape cell death and to proliferate in hypoxic environments. Our data illustrates that Carbonic Anhydrases IX and XII (CA IX/XII) are critical for leukaemic cell survival in the O2 -deprived milieu. CA IX and XII function as transmembrane proteins that mediate intracellular pH under low O2 conditions. Because maintaining a neutral pH represents a key survival mechanism for tumour cells in O2 -deprived settings, we sought to elucidate the role of dual CA IX/XII inhibition as a novel strategy to eliminate AML cells under hypoxic conditions. Our findings demonstrate that the dual CA IX/XII inhibitor FC531 may prove to be of value as an adjunct to chemotherapy for the treatment of AML.