Browsing by Author "Temm, Constance J."

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    Aberrant epigenetic and transcriptional events associated with breast cancer risk
    (BMC, 2022-02-09) Marino, Natascia; German, Rana; Podicheti, Ram; Rusch, Douglas B.; Rockey, Pam; Huang, Jie; Sandusky, George E.; Temm, Constance J.; Althouse, Sandra; Nephew, Kenneth P.; Nakshatri, Harikrishna; Liu, Jun; Vode, Ashley; Cao, Sha; Storniolo, Anna Maria V.; Medicine, School of Medicine
    Background: Genome-wide association studies have identified several breast cancer susceptibility loci. However, biomarkers for risk assessment are still missing. Here, we investigated cancer-related molecular changes detected in tissues from women at high risk for breast cancer prior to disease manifestation. Disease-free breast tissue cores donated by healthy women (N = 146, median age = 39 years) were processed for both methylome (MethylCap) and transcriptome (Illumina's HiSeq4000) sequencing. Analysis of tissue microarray and primary breast epithelial cells was used to confirm gene expression dysregulation. Results: Transcriptomic analysis identified 69 differentially expressed genes between women at high and those at average risk of breast cancer (Tyrer-Cuzick model) at FDR < 0.05 and fold change ≥ 2. Majority of the identified genes were involved in DNA damage checkpoint, cell cycle, and cell adhesion. Two genes, FAM83A and NEK2, were overexpressed in tissue sections (FDR < 0.01) and primary epithelial cells (p < 0.05) from high-risk breasts. Moreover, 1698 DNA methylation changes were identified in high-risk breast tissues (FDR < 0.05), partially overlapped with cancer-related signatures, and correlated with transcriptional changes (p < 0.05, r ≤ 0.5). Finally, among the participants, 35 women donated breast biopsies at two time points, and age-related molecular alterations enhanced in high-risk subjects were identified. Conclusions: Normal breast tissue from women at high risk of breast cancer bears molecular aberrations that may contribute to breast cancer susceptibility. This study is the first molecular characterization of the true normal breast tissues, and provides an opportunity to investigate molecular markers of breast cancer risk, which may lead to new preventive approaches.
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    Adenoviral Vectors Expressing Human Endostatin–Angiostatin and Soluble Tie2: Enhanced Suppression of Tumor Growth and Antiangiogenic Effects in a Prostate Tumor Model
    (Elsevier, 2005-12-01) Raikwar, Sudhanshu P.; Temm, Constance J.; Raikwar, Nandita S.; Kao, Chinghai; Molitoris, Bruce A.; Gardner, Thomas A.; Urology, School of Medicine
    Angiogenesis is essential for prostate cancer development and metastasis. Antiangiogenic therapy targeting tumor neovasculature, therefore, represents a promising approach for prostate cancer treatment. We hypothesized that adenoviral-mediated delivery of a combination of antiangiogenic factors might have an enhanced antitumor response. We developed the adenoviral vectors Ad-hEndo-angio, expressing a unique, chimeric human endostatin–angiostatin fusion protein, and Ad-sTie2, expressing a soluble form of endothelium-specific receptor tyrosine kinase Tie2. Matrigel angiogenesis assays using Ad-hEndo-angio revealed significant inhibition of tubular network formation and endothelial sprouting compared to Ad-sTie2. In vivo studies in a bilateral PC-3 tumor xenograft model following either intratumoral or systemic administration of Ad-hEndo-angio led to enhanced tumor growth suppression compared to Ad-sTie2. A novel finding is that an intratumoral, combination therapy employing one-half the dose of Ad-hEndo-angio as well as Ad-sTie2 led to a complete regression of the injected, as well as the contralateral uninjected, tumor and prolonged the tumor-free survival in 80% of the animals. In addition, a novel, real-time, intravital imaging modality was used to monitor antiangiogenic responses following adenoviral-mediated gene transfer. These results suggest that a combinatorial antiangiogenic gene therapy approach involving Ad-hEndo-angio and Ad-sTie2 could become a novel form of treatment for localized human prostate cancer.
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    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 Medicine
    Breast 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.
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    BreastDefend enhances effect of tamoxifen in estrogen receptor-positive human breast cancer in vitro and in vivo
    (BioMed Central, 2017-02-16) Cheng, Shujie; Castillo, Victor; Welty, Matt; Alvarado, Mark; Eliaz, Isaac; Temm, Constance J.; Sandusky, George E.; Sliva, Daniel; Department of Pathology and Laboratory Medicine, IU School of Medicine
    BACKGROUND: Tamoxifen (TAM) has been widely used for the treatment of estrogen receptor (ER)-positive breast cancer and its combination with other therapies is being actively investigated as a way to increase efficacy and decrease side effects. Here, we evaluate the therapeutic potential of co-treatment with TAM and BreastDefend (BD), a dietary supplement formula, in ER-positive human breast cancer. METHODS: Cell proliferation and apoptosis were determined in ER-positive human breast cancer cells MCF-7 by MTT assay, quantitation of cytoplasmic histone-associated DNA fragments and expression of cleaved PARP, respectively. The molecular mechanism was identified using RNA microarray analysis and western blotting. Tumor tissues from xenograft mouse model were analyzed by immunohistochemistry. RESULTS: Our data clearly demonstrate that a combination of 4-hydroxytamoxifen (4-OHT) with BD lead to profound inhibition of cell proliferation and induction of apoptosis in MCF-7 cells. This effect is consistent with the regulation of apoptotic and TAM resistant genes at the transcription and translation levels. Importantly, TAM and BD co-treatment significantly enhanced apoptosis, suppressed tumor growth and reduced tumor weight in a xenograft model of human ER-positive breast cancer. CONCLUSION: BD sensitized ER-positive human breast cancer cells to 4-OHT/TAM treatment in vitro and in vivo. BreastDefend can be used in an adjuvant therapy to increase the therapeutic effect of tamoxifen in patients with ER-positive breast cancer.
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    Clinical, histopathologic and molecular features of idiopathic and diabetic nodular mesangial sclerosis in humans
    (Oxford University Press, 2021) Eadon, Michael T.; Lampe, Sam; Baig, Mirza M.; Collins, Kimberly S.; Ferreira, Ricardo Melo; Mang, Henry; Cheng, Ying-Hua; Barwinska, Daria; El-Achkar, Tarek M.; Schwantes-An, Tae-Hwi; Winfree, Seth; Temm, Constance J.; Ferkowicz, Michael J.; Dunn, Kenneth W.; Kelly, Katherine J.; Sutton, Timothy A.; Moe, Sharon M.; Moorthi, Ranjani N.; Phillips, Carrie L.; Dagher, Pierre C.; Medicine, School of Medicine
    Background: Idiopathic nodular mesangial sclerosis, also called idiopathic nodular glomerulosclerosis (ING), is a rare clinical entity with an unclear pathogenesis. The hallmark of this disease is the presence of nodular mesangial sclerosis on histology without clinical evidence of diabetes mellitus or other predisposing diagnoses. To achieve insights into its pathogenesis, we queried the clinical, histopathologic and transcriptomic features of ING and nodular diabetic nephropathy (DN). Methods: All renal biopsy reports accessioned at Indiana University Health from 2001 to 2016 were reviewed to identify 48 ING cases. Clinical and histopathologic features were compared between individuals with ING and DN (n = 751). Glomeruli of ING (n = 5), DN (n = 18) and reference (REF) nephrectomy (n = 9) samples were isolated by laser microdissection and RNA was sequenced. Immunohistochemistry of proline-rich 36 (PRR36) protein was performed. Results: ING subjects were frequently hypertensive (95.8%) with a smoking history (66.7%). ING subjects were older, had lower proteinuria and had less hyaline arteriolosclerosis than DN subjects. Butanoate metabolism was an enriched pathway in ING samples compared with either REF or DN samples. The top differentially expressed gene, PRR36, had increased expression in glomeruli 248-fold [false discovery rate (FDR) P = 5.93 × 10-6] compared with the REF and increased 109-fold (FDR P = 1.85 × 10-6) compared with DN samples. Immunohistochemistry revealed a reduced proportion of cells with perinuclear reaction in ING samples as compared to DN. Conclusions: Despite similar clinical and histopathologic characteristics in ING and DN, the uncovered transcriptomic signature suggests that ING has distinct molecular features from nodular DN. Further study is warranted to understand these relationships.
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    Critical role of phosphorylation of serine 165 of YBX1 on the activation of NF- B in colon cancer
    (Office of the Vice Chancellor for Research, 2015-04-17) Prabhu, Lakshmi; Mundade, Rasika; Wang, Benlian; Wei, Han; Hartley, Antja-Voy; McElyea, Kyle; Temm, Constance J.; Sandusky, George; Liu, Yunlong; Lu, Tao
    Y-box binding protein 1 (YBX1) is a multifunctional protein known to facilitate many of the hallmarks of cancer. Elevated levels of YBX1 protein are highly correlated with cancer progression, making it an excellent marker in cancer. The connection between YBX1 and the important nuclear factor B (NF-B), has never been previously reported. Here, we show that overexpression of wild type YBX1 (wtYBX1) activates NF-B, suggesting that YBX1 is a potential NF-B activator. Furthermore, using mass spectrometry analysis, we identified novel phosphorylation of serine 165 (S165) on YBX1. Overexpression of the S165A-YBX1 mutant in either 293 cells or colon cancer HT29 cells showed dramatically reduced NF-B activating ability as compared to that of wtYBX1, confirming that S165 phosphorylation is critical for the activation of NF-B by YBX1. We further show that expression of the S165A-YBX1 mutant dramatically decreased the expression of NF-B-inducible genes, reduced cell growth, and compromised tumorigenic ability as compared to wtYBX1. Taken together, we provide the first evidence that YBX1 functions as a tumor promoter via NF-B activation, and phosphorylation of S165 of YBX1 is critical for this function. Therefore, our important discovery may lead to blocking S165 phosphorylation as a potential therapeutic strategy to treat colon cancer.
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    Critical role of phosphorylation of serine 165 of YBX1 on the activation of NF-κB in colon cancer.
    (Impact Journals, 2015-10-06) Prabhu, Lakshmi; Mundade, Rasika; Wang, Benlian; Wei, Han; Hartley, Antja-Voy; Martin, Matthew; McElyea, Kyle; Temm, Constance J.; Sandusky, George; Liu, Yunlong; Lu, Tao; Department of Pharmacology and Toxicology, IU School of Medicine
    Y-box binding protein 1 [YBX1] is a multifunctional protein known to facilitate many of the hallmarks of cancer. Elevated levels of YBX1 protein are highly correlated with cancer progression, making it an excellent marker in cancer. The connection between YBX1 and the important nuclear factor κB [NF-κB] has never been reported. Here, we show that overexpression of wild type YBX1 [WT-YBX1] activates NF-κB, suggesting that YBX1 is a potential NF-κB activator. Furthermore, using mass spectrometry analysis we identified novel phosphorylation of serine 165 [S165] on YBX1. Overexpression of the S165A-YBX1 mutant in either HEK293 cells or colon cancer HT29 cells showed dramatically reduced NF-κB activating ability as compared with that of WT-YBX1, confirming that S165 phosphorylation is critical for the activation of NF-κB by YBX1. We also show that expression of the S165A-YBX1 mutant dramatically decreased the expression of
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    FAM83A is a potential biomarker for breast cancer initiation
    (Springer, 2022-02-19) Marino, Natascia; German, Rana; Podicheti, Ram; Rockey, Pam; Sandusky, George E.; Temm, Constance J.; Nakshatri, Harikrishna; Addison, Rebekah J.; Selman, Bryce; Althouse, Sandra K.; Storniolo , Anna Maria V.; Medicine, School of Medicine
    Background Family with sequence similarity 83 member A (FAM83A) presents oncogenic properties in several cancers including breast cancer. Recently, we reported FAM83A overexpression in normal breast tissues from women at high risk of breast cancer. We now hypothesize that FAM83A is a key factor in breast cancer initiation. Methods Immunohistochemical staining was used to evaluate FAM83A protein levels in both a normal breast tissue microarray (TMA, N = 411) and a breast tumor TMA (N = 349). EGFR staining and its correlation with FAM83A expression were also assessed. Lentivirus-mediated manipulation of FAM83A expression in primary and hTERT-immortalized breast epithelial cells was employed. Biological and molecular alterations upon FAM83A overexpression/downregulation and FAM83A’s interaction partners were investigated. Results TMA analysis revealed a 1.5-fold increase in FAM83A expression level in breast cancer cases as compared with normal breast tissues (p < 0.0001). FAM83A protein expression was directly correlated with EGFR level in both normal and breast cancer tissues. In in vitro assays, exogenous expression of FAM83A in either primary or immortalized breast epithelial cells promoted cell viability and proliferation. Additionally, Ingenuity Pathway Analysis (IPA) revealed that FAM83A overexpression in primary cells affected the expression of genes involved in cellular morphology and metabolism. Mass spectrometry analysis identified DDX3X and LAMB3 as potential FAM83A interaction partners in primary cells, while we detected FAM83A interaction with cytoskeleton reorganization factors, including LIMA1, MYH10, PLEC, MYL6 in the immortalized cells. Conclusions This study shows that FAM83A promotes metabolic activation in primary breast epithelial cells and cell proliferation in both primary and immortalized cells. These findings support its role in early breast oncogenesis.
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    Hypoxia-Inducible Factor-1α Regulates CD55 in Airway Epithelium
    (American Thoracic Society, 2016-12) Pandya, Pankita H.; Fisher, Amanda J.; Mickler, Elizabeth A.; Temm, Constance J.; Lipking, Kelsey P.; Gracon, Adam; Rothhaar, Katia; Sandusky, George E.; Murray, Mary; Pollok, Karen; Renbarger, Jamie; Blum, Janice S.; Lahm, Tim; Wilkes, David S.; Microbiology and Immunology, School of Medicine
    Airway epithelial CD55 down-regulation occurs in several hypoxia-associated pulmonary diseases, but the mechanism is unknown. Using in vivo and in vitro assays of pharmacologic inhibition and gene silencing, the current study investigated the role of hypoxia-inducible factor (HIF)-1α in regulating airway epithelial CD55 expression. Hypoxia down-regulated CD55 expression on small-airway epithelial cells in vitro, and in murine lungs in vivo; the latter was associated with local complement activation. Treatment with pharmacologic inhibition or silencing of HIF-1α during hypoxia-recovered CD55 expression in small-airway epithelial cells. HIF-1α overexpression or blockade, in vitro or in vivo, down-regulated CD55 expression. Collectively, these data show a key role for HIF-1α in regulating the expression of CD55 on airway epithelium.
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    IMMUNOHISTOCHEMISTRY EXPRESSION OF KLOTHO IN BONE MARROW BIOPSIES FROM NORMAL, MGUS, AND PLASMA CELL MYELOMA
    (Office of the Vice Chancellor for Research, 2012-04-13) Parker, Jamie; Temm, Constance J.; Chirgwin, Jon; Suvannasankha, Attaya; Imel, Erik; Sandusky, George
    Klotho is an anti-aging gene, which has been shown to inhibit the insulin and insulin-like growth factor 1 (IGF-1) pathways in mice hepatocytes and myocytes. Immunochemistry analysis of Klotho expression in breast tissue arrays revealed high expression in normal breast, but very low expression in breast cancer. In this study we examined eight normal bone marrow, eight MGUS (monoclonal gammopathy of undetermined significance), and forty-two cases of plasma cell myeloma by immunohistochemistry with the Klotho antibody. The immunostaining of the Klotho antibody was localized in the cyto-plasm and as punctate granular staining of myeloma cells in the marrow. In the accompanying bone marrow clots, Klotho was seen as strong punctate granules on myeloma cells and not on other peripheral white blood cells. There was no staining of plasma cells in the eight normal bone marrow cas-es. Slight cytoplasmic staining was seen in myeloid series of cells in the normal bone marrow and in megakaryocytes. In the eight MGUS cases, there was very minimal cytoplasmic staining in a few of the myeloma cells. Minimal staining was seen in the myeloid series of cells in the marrow in these cases. Klotho was highly expressed in the myeloma cases and no staining in the normal and MGUS cases. In conclusion, Klotho was highly expressed in patients with myeloma in myelomas cells in the bone marrow. This project was sponsored by the Life Health Science Internship Program