Browsing by Subject "breast cancer"
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Item A Role for Adipocytes and Adipose Stem Cells in the Breast Tumor Microenvironment and Regenerative Medicine(Frontiers Media S.A., 2021-11-29) Brock, Courtney K.; Hebert, Katherine L.; Artiles, Maria; Wright, Maryl K.; Cheng, Thomas; Windsor, Gabreille O.; Nguyen, Khao; Alzoubi, Madlin S.; Collins-Burow, Bridgette M.; Martin, Elizabeth C.; Lau, Frank H.; Bunnell, Bruce A.; Burow, Matthew E.Obesity rates are climbing, representing a confounding and contributing factor to many disease states, including cancer. With respect to breast cancer, obesity plays a prominent role in the etiology of this disease, with certain subtypes such as triple-negative breast cancer having a strong correlation between obesity and poor outcomes. Therefore, it is critical to examine the obesity-related alterations to the normal stroma and the tumor microenvironment (TME). Adipocytes and adipose stem cells (ASCs) are major components of breast tissue stroma that have essential functions in both physiological and pathological states, including energy storage and metabolic homeostasis, physical support of breast epithelial cells, and directing inflammatory and wound healing responses through secreted factors. However, these processes can become dysregulated in both metabolic disorders, such as obesity and also in the context of breast cancer. Given the well-established obesity-neoplasia axis, it is critical to understand how interactions between different cell types in the tumor microenvironment, including adipocytes and ASCs, govern carcinogenesis, tumorigenesis, and ultimately metastasis. ASCs and adipocytes have multifactorial roles in cancer progression; however, due to the plastic nature of these cells, they also have a role in regenerative medicine, making them promising tools for tissue engineering. At the physiological level, the interactions between obesity and breast cancer have been examined; here, we will delineate the mechanisms that regulate ASCs and adipocytes in these different contexts through interactions between cancer cells, immune cells, and other cell types present in the tumor microenvironment. We will define the current state of understanding of how adipocytes and ASCs contribute to tumor progression through their role in the tumor microenvironment and how this is altered in the context of obesity. We will also introduce recent developments in utilizing adipocytes and ASCs in novel approaches to breast reconstruction and regenerative medicine.Item Community Profile Report of Breast Health: Greater Fort Worth Affiliate(Greater Fort Worth Affiliate of Susan G. Komen for the Cure, 2011) Lurie, Sue G.The purpose of the Community Profile report is to gain and present current information on the breast health of communities in the Greater Fort Worth service area. This information is collected to identify and assess local priorities for breast cancer education, screening, treatment and treatment support services (health and social support services) currently provided and needed for the under and uninsured population at greatest risk of breast cancer. The information gathered from the periodic Community Profile effort is used to establish Grant funding priorities and to direct Affiliate Strategic planning with the desired result of effective use and distribution of Affiliate funding and resources for breast health needs.Item Community Profile Report of Breast Health: North Texas Affiliate(North Texas Affiliate of Susan G. Komen for the Cure, 2011) Lurie, Sue G.The purpose of the Community Profile assessment conducted by the North Texas Affiliate of Susan G. Komen for the Cure for 2011 is to gain and present current information on the health of communities within the North Texas region. This information was collected to identify and assess local priorities for health education, screening, health and social support services that are currently provided, and those that are needed for the population at the greatest risk of breast cancer.Item Community Profile Report of Breast Health: Tarrant County Affiliate(Susan G. Komen for the Cure Tarrant County Affiliate, 2009) Lurie, Sue G.The purpose of this Community Profile (Needs Assessment) is to gain information on the breast health knowledge of the community and of services provided for the Tarrant County population with the greatest risk of breast cancer.Item Cytotoxic Activity of the Mesoionic Compound MIH 2.4Bl in Breast Cancer Cell Lines(Sage Publications, 2020-07-01) Amaral de Mascena Costa, Luciana; Harmon, Ashlyn C.; Aguiar Coelho Teixeira, Alvaro; Cassio Silva de Lima, Filipe; de Sousa Araujo, Silvany; Del Piero, Fabio; Diogenes da Silva Souza, Helivaldo; Filgueiras de Athayde Filho, Petronio; Alves Junior, Severino; de Mascena Diniz Maia, Maria; Wischral, Aurea; Adriao Gomes Filho, Manoel; Mathis, J. MichaelIn this work, we report the synthesis of a new 1,3-thiazolium-5-thiolate derivative of a mesoionic compound (MIH 2.4Bl) and the characterization of its selective cytotoxicity on a panel of breast cancer cells lines. The cytotoxic effect of MIH 2.4Bl on breast cancer cell lines was determined by XTT and crystal violet assays, flow cytometry analysis, electron microscopy characterization, and terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) apoptosis assays. As determined using XTT cell growth and survival assays, MIH 2.4Bl exhibited growth inhibition activity on most breast cancer cell lines tested, compared with normal human mammary epithelial cells. Three breast cancer cell lines (MCF-7, T-47D, and ZR-75-1) showed a more potent sensitivity index to growth inhibition by MIH 2.4Bl than the other breast cancer cell lines. Interestingly, these 3 cell lines were derived from tumors of Luminal A origin and have ER (estrogen receptor), PR (progesterone receptor), and HER2 (human epidermal growth factor receptor 2) positive expression. Additional analysis of cytotoxicity mediated by MIH 2.4Bl was performed using the MCF-7 cell line. MCF-7 cells displayed both time- and dose-dependent decreases in cell growth and survival, with a maximum cytotoxic effect observed at 72 and 96 hours. The MCF-7 cells were also characterized for cell cycle changes upon treatment with MIH 2.4Bl. Using flow cytometry analysis of cell cycle distribution, a treatment-dependent effect was observed; treatment of cells with MIH 2.4Bl increased the G2/M population to 34.2% compared with 0.1% in untreated (control) cells. Ultrastructural analysis of MFC-7 cells treated with MIH 2.4Bl at 2 different concentrations (37.5 and 75 muM) was performed by transmission electron microscopy. Cells treated with 37.5 muM MIH 2.4Bl showed morphologic changes beginning at 6 hours after treatment, while cells treated with 75 muM showed changes beginning at 3 hours after treatment. These changes were characterized by an alteration of nuclear morphology and mitochondrial degeneration consistent with apoptotic cell death. Results of a TUNEL assay performed on cells treated for 96 hours with MIH 2.4Bl supported the observation of apoptosis. Together, these results suggest that MIH 2.4Bl is a promising candidate for treating breast cancer and support further in vitro and in vivo investigation.Item Distinct Roles of mTOR Targets S6K1 and S6K2 in Breast Cancer(MDPI, 2020-02-11) Sridharan, Savitha; Basu, AlakanandaThe mechanistic target of rapamycin (mTOR) is a master regulator of protein translation, metabolism, cell growth and proliferation. It forms two complexes, mTOR complex 1 (mTORC1) and 2 (mTORC2). mTORC1 is frequently deregulated in many cancers, including breast cancer, and is an important target for cancer therapy. The immunosuppressant drug rapamycin and its analogs that inhibit mTOR are currently being evaluated for their potential as anti-cancer agents, albeit with limited efficacy. mTORC1 mediates its function via its downstream targets 40S ribosomal S6 kinases (S6K) and eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). There are two homologs of S6K: S6K1 and S6K2. Most of the earlier studies focused on S6K1 rather than S6K2. Because of their high degree of structural homology, it was generally believed that they behave similarly. Recent studies suggest that while they may share some functions, they may also exhibit distinct or even opposite functions. Both homologs have been implicated in breast cancer, although how they contribute to breast cancer may differ. The purpose of this review article is to compare and contrast the expression, structure, regulation and function of these two S6K homologs in breast cancer.Item Effect of CRISPR MIEN1 Knockout in Metastatic Breast Cancer Cells(2018-12-01) Van Treuren, Timothy R.; Vishwanatha, Jamboor K.; Basu, Alakananda; Basha, RiyazMigration and Invasion Enhancer 1 (MIEN1) is an oncogene which is involved in facilitating the migration and invasion of cancer cells through actin dynamics and gene expression. Increased MIEN1 expression in many types of tumors correlates with disease progression and metastatic propensity. The precise mechanism by which MIEN1 functions is yet to be understood. The goal of these studies is to progress toward determination of the mechanisms and genetic context in which MIEN1 functions contribute to cancer progression. It was hypothesized that Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) mediated knockout of MIEN1 in metastatic breast cancer cells would result in reduced migration and invasion. CRISPR genome editing effectively produced specific genomic deletions in the MIEN1 gene which led to the elimination of its expression in these breast cancer cells. Migration in MDA-MB-231 (231) MIEN1 knockout (MIEN1-KO) cells exhibited no difference when compared to parental 231, which was in contrast with previous siRNA studies. Signaling in several MIEN1-KO pools was inconsistent. Knocking out MIEN1 in 231 derivative cell lines showed few significant alterations in the growth, migration, invasion, signaling, despite significant changes in metabolism. However, re-expression of the MIEN1 protein containing a mutant immunoreceptor tyrosine-based activation motif (ITAM) domain resulted in significantly decreased invasion. This revealed that MIEN1-KO 231 derivative cells were susceptible to interference of compensatory mechanisms and demonstrates the importance of the migration and invasion pathways in which MIEN1 participates in breast cancer metastasis. These findings also suggest MIEN1 may still be a promising therapeutic target to inhibit metastasis if inhibitors can be developed which block ITAM function without affecting localization or expression.Item Effect of CRISPR MIEN1 knockout in metastatic breast cancer cells(2018-12) Van Treuren, Timothy; Vishwanatha, Jamboor K.; Basu, Alakananda; Basha, RiyazMigration and Invasion Enhancer 1 (MIEN1) is an oncogene which is involved in facilitating the migration and invasion of cancer cells through actin dynamics and gene expression. Increased MIEN1 expression in many types of tumors correlates with disease progression and metastatic propensity. The precise mechanism by which MIEN1 functions is yet to be understood. The goal of these studies is to progress toward determination of the mechanisms and genetic context in which MIEN1 functions contribute to cancer progression. It was hypothesized that Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) mediated knockout of MIEN1 in metastatic breast cancer cells would result in reduced migration and invasion. CRISPR genome editing effectively produced specific genomic deletions in the MIEN1 gene which led to the elimination of its expression in these breast cancer cells. Migration in MDA-MB-231 (231) MIEN1 knockout (MIEN1-KO) cells exhibited no difference when compared to parental 231, which was in contrast with previous siRNA studies. Signaling in several MIEN1-KO pools was inconsistent. Knocking out MIEN1 in 231 derivative cell lines showed few significant alterations in the growth, migration, invasion, signaling, despite significant changes in metabolism. However, re-expression of the MIEN1 protein containing a mutant immunoreceptor tyrosine-based activation motif (ITAM) domain resulted in significantly decreased invasion. This revealed that MIEN1-KO 231 derivative cells were susceptible to interference of compensatory mechanisms and demonstrates the importance of the migration and invasion pathways in which MIEN1 participates in breast cancer metastasis. These findings also suggest MIEN1 may still be a promising therapeutic target to inhibit metastasis if inhibitors can be developed which block ITAM function without affecting localization or expression.Item EGCG and Its Role in Prostate Cancer Angiogenesis(2005-05-01) Thomas, Rusha; Porunelloor Mathew; Ming-Chi Wu; Dan DimitrijevichThomas, Rusha, EGCG and its role in prostate cancer angiogenesis. Master of Science (Biochemistry and Molecular Biology), May 2005, 47 pages, 14 illustrations, reference list, 44 titles. Hypoxia inducible factor-1 (HIF-1)-mediated upregulation of vascular endothelial growth factor (VEGF) has been implicated in angiogenesis associated with malignancies. HIF-1 consists of a constitutively expressed HIF-1β subunit, and a hypoxia-inducible HIF-1α subunit. Hypoxic induction of HIF-1α correlates with increased transcriptional activation of its downstream target genes, including VEGF. Epidemiologic and laboratory studies indicate that green tea has cancer preventive activity which has been attributed to its polyphenol components, the major one being epigallocatechin gallate (EGCG). This study investigated the effect of EGCG on normoxic VEGF expression in PC-3ML human prostate cancer cells. In contrast to previous studies where EGCG inhibited VEGF expression in breast and colon cancer cell lines, our results demonstrated that EGCG has the ability to upregulate HIF-1α transcription factor via inhibition of prolyl hydroxylation and subsequent von Hippel-Lindau protein interaction. HIF-1α upregulation by EGCG led to increased VEGF promoter activity and protein expression.Item Formulation, characterization and validation of CpG functionalized PLGA bacteriomimetic nanoparticles for breast cancer immunotherapy(2015-05-01) Kokate, Rutika A.; Harlan P. JonesTargeted Immunotherapy represents a potential and innovative means to combat cancer. Cancer vaccines designed against a specific tumor antigen have been efficiently utilized to trigger immune responses against tumor cells. Despite the preliminary evidence in animal models, low immunogenicity is one of the major hurdles in the development of vaccines in humans. Several approaches including the use of an “ideal” tumor antigen, appropriate delivery techniques, immune boosting strategies with co-stimulatory molecules are being explored to surmount this obstacle. The goal of this dissertation project was to utilize polymeric nanoparticles (NPs) as a vehicle to deliver Tumor Associated Antigen (TAA) that would elicit a strong antitumor immune response. In the present study, we successfully formulated CpG surface functionalized Tag encapsulating PLGA nanoparticles (CpG-NP-Tag) and tested their efficacy using in vivo and ex vivo experimental models. Specifically, we developed and characterized NPs for physicochemical properties including particle size, surface charge, surface morphology, Polydispersity index (PDI), encapsulation efficiency and CpG ligand binding efficiency. CpGNP-Tag NPs were found be of desired size (220-230 nm) and surface charge (negative zeta potential). Particles were non agglomerated, spherical in shape and uniform in size with PDI in the range of 0.03-0.1. Due to the hydrophobic nature of the encapsulated entity (Tag), the encapsulation efficiency was limited to 30-40%. CpG ligand conjugation on the surface of NPs was confirmed using Fluorescence Correlation Spectroscopy (FCS). CpG ligand binding efficiency was found to be around 10-14%. We also found that CpG-NP-Tag NP formulation had desired properties (size, charge and morphology) for efficient uptake by phagocytic antigen presenting cells (APCs) such as dendritic cells (DCs). The major aim of our studies was to test the antitumor efficacy of NPs. Using a prophylactic syngenic Balb/c mice model, we demonstrated that CpG-NP-Tag can serve as an efficient tool to bolster antitumor immunity and thus could be used as a platform for the development of NP based immunotherapeutic interventions in future. We found that CpG-NPTag NP immunization attenuated tumor growth, proliferation, angiogenesis and induced apoptotic tumor cell death. These NPs indicated immunostimulatory potential by enhancing tumor CD4+ and CD8+ T cell infiltration as well as local IFN-γ production. Overall, from these in vivo studies we concluded that CpG-NP-Tag promotes IFN-γ secretion which possibly mediates the inhibited tumor growth, angiogenesis and enhanced T cell mediated immunity which facilitates tumor cell death via apoptosis. To understand the mechanism by which CpG-NP-Tag imparts antitumor effects we used ex vivo model of APCs. Studies were conducting using Bone Marrow Derived Dendritic Cells (BMDCs) isolated from female Balb/c mice. We demonstrated enhanced NP uptake, preferential Endosomal localization, and increased population of CD80/86 expressing BMDCs in case of CpG-NP-Tag pulsed BMDCs indicating these NPs could serve as candidates for DC based vaccines in future. In summary, both ex vivo and in vivo studies conducted with CpG-NP-Tag NPs provide insight in the development of particulate vaccines in cancer immunotherapy.Item Genetic characterization of comorbidity patterns in aging associated diseases using integrative genomics(2019-08) Pathak, Gita A.; Phillips, Nicole R.; Planz, John V.; Barber, Robert C.; Zhou, Zhengyang; Gryczynski, IgnacyThe aging population in the US continues to grow at an exponential rate estimated to reach more than 90 million by 2060. The coexistence of two or more diseases (comorbidity) is prevalent in ages 65 years and above, and the number of comorbidities increases with age. The genetic factors underlying presence and absence of comorbidities is a severely understudied research domain. Alzheimer's disease (AD) is a type of dementia affecting 5.5 million people with an average age of diagnosis at 70 years. Hypertension is a coexisting condition in 60% AD individuals, also known as direct comorbidity. On the other hand, cancer is reported to be inversely comorbid with AD; individuals with cancer history have been reported to have lower risk of AD and vice versa. Furthermore, individuals with cancer history are diagnosed with long term side effects of radiation therapy — radiotoxicity. Twin-based studies have reported that certain gene variants are associated with radiotoxicity phenotypes with a heritability of 66%. This study proposes to investigate genetic factors associated with the direct and inverse comorbidity of AD with hypertension and cancer, and proctitis — a radiotoxicity phenotype observed in survivors of prostate cancer. The study aims to integrate gene variants, derived-gene expression and copy number variation (CNV), followed by functional and pathway-based prioritization of observed findings. We used genome-wide and cerebral spinal fluid profile to investigate presence of hypertension with AD to evaluate individual-level differences, followed by targeted investigation of neighboring gene expression profiles of identified variants. We found several novel genes associated with AD-hypertension comorbidity. The investigation between AD and cancer identified regions in chromosomes 4, 5 and 19 that are targeted by miRNA-17 family along with other miRNAs reported to be inversely expressed and play opposite role in pathogenicity of both diseases. The SNP-derived transcriptomic profile between AD and cancer highlighted involvement of sirtuin signaling. The findings together indicate involvement of mitochondrial and metabolic dysregulation which possibly contribute in differences of the epithelial-mesenchymal-transition. The SNP-derived expression and CNV association with proctitis highlighted genes involved in DNA-repair and mitochondrial ROS damage pathways.Item Identification of novel alternative splicing biomarkers for breast cancer with LC/MS/MS and RNA-Seq(BioMed Central Ltd., 2020-12-03) Zhang, Fan; Deng, Chris K.; Wang, Mu; Deng, Bin; Barber, Robert C.; Huang, GangBackground: Alternative splicing isoforms have been reported as a new and robust class of diagnostic biomarkers. Over 95% of human genes are estimated to be alternatively spliced as a powerful means of producing functionally diverse proteins from a single gene. The emergence of next-generation sequencing technologies, especially RNA-seq, provides novel insights into large-scale detection and analysis of alternative splicing at the transcriptional level. Advances in Proteomic Technologies such as liquid chromatography coupled tandem mass spectrometry (LC-MS/MS), have shown tremendous power for the parallel characterization of large amount of proteins in biological samples. Although poor correspondence has been generally found from previous qualitative comparative analysis between proteomics and microarray data, significantly higher degrees of correlation have been observed at the level of exon. Combining protein and RNA data by searching LC-MS/MS data against a customized protein database from RNA-Seq may produce a subset of alternatively spliced protein isoform candidates that have higher confidence. Results: We developed a bioinformatics workflow to discover alternative splicing biomarkers from LC-MS/MS using RNA-Seq. First, we retrieved high confident, novel alternative splicing biomarkers from the breast cancer RNA-Seq database. Then, we translated these sequences into in silico Isoform Junction Peptides, and created a customized alternative splicing database for MS searching. Lastly, we ran the Open Mass spectrometry Search Algorithm against the customized alternative splicing database with breast cancer plasma proteome. Twenty six alternative splicing biomarker peptides with one single intron event and one exon skipping event were identified. Further interpretation of biological pathways with our Integrated Pathway Analysis Database showed that these 26 peptides are associated with Cancer, Signaling, Metabolism, Regulation, Immune System and Hemostasis pathways, which are consistent with the 256 alternative splicing biomarkers from the RNA-Seq. Conclusions: This paper presents a bioinformatics workflow for using RNA-seq data to discover novel alternative splicing biomarkers from the breast cancer proteome. As a complement to synthetic alternative splicing database technique for alternative splicing identification, this method combines the advantages of two platforms: mass spectrometry and next generation sequencing and can help identify potentially highly sample-specific alternative splicing isoform biomarkers at early-stage of cancer.Item Involvement of S6 Kinase in Breast Cancer(2013-12-01) Sridharan, Savitha; Basu, AlakanandaSridharan, S., Involvement of S6 Kinase in Breast Cancer. Doctor of Philosophy (Cancer Biology), November 2013, 129pp, 19 illustrations, 215 references. The 40S ribosomal protein S6 Kinase (S6K) is activated downstream of the mammalian target of rapamycin (mTOR)and is believed to play and important role in protein translation. In mammalian cells S6K is represented by two highly homologous proteins, S6K1 and S6K2. Both homologs have been shown to be amplicfied and over expressed in breast cancer cells and tissues. While the regulation and functions of S6K1 have been addressed, little is known about those of S6K2 . Hence we sought to examine the causes and consequences of elevated S6K2 levels in breast cancer cells. While the depletion of S6K1 decreased breast cancer cell death, silencing of S6K2 substantially increased it in response to apoptotic and chemotherapeutic agents. We then explored the mechanism by which S6K2 mediates survival and observed that in contrast to S6K1, S6K2 depletion decreased the activation of the prosurvival protein Akt and increased the level of proapoptotic proteins p53 and bid. Following this observation, we sought to determine the pathways(s) contributing to the overexpression of S6K2 in breast cancer cells. Due to its role as a prognostic indicator in estrogen receptor (ER) – positive tumors, we studied the role of the estrogen signaling pathways in regulating S6K2 levels. Estradiol and estrogen receptor alpha (ERα) positively regulated S6K2 protein but did not affect its mRNA levels, suggesting post-transcriptional regulation. We further observed that S6K2 regulated cell survival downstream of estrogen in ER-positive breast cancer cells. These findings strongly suggest that S6K2 is critical for the survival of breast cancer cells and that targeting S6K2 in combination with chemotherapeutic agents is a novel strategy to promote breast cancer cell death.Item Lectin-like transcript 1 as a natural killer cell-mediated immunotherapeutic target for triple negative breast cancer and prostate cancer(OAE Publishing, Inc., 2019-12-17) Sun, Yuanhong; Malaer, Joseph D.; Mathew, Porunelloor A.Breast and prostate cancer are the leading causes of death in females and males, respectively. Triple negative breast cancer (TNBC) does not express the estrogen receptor, progesterone receptor, or human epidermal growth factor receptor 2, resulting in limited treatment options. Androgen deprivation therapy is the standard care for prostate cancer patients; however, metastasis and recurrence are seen in androgen-independent prostate cancer. Both prostate and breast cancer show higher resistance after recurrence and metastasis, which increases the difficulty of treatment. Natural killer (NK) cells play a critical role during innate immunity and tumor recognition and elimination. NK cell function is determined by a delicate balance of inhibitory signals and activation signals received through cell surface receptors. Lectin-like transcript 1 (LLT1, CLEC2D, OCIL) is a ligand of NK cell inhibitory receptor NKRP1A (CD161). Several studies have that reported higher expression of LLT1 is associated with the development of various tumors. Our studies revealed that TNBC and prostate cancer cells express higher levels of LLT1. In the presence of a monoclonal antibody against LLT1, NK cell-mediated killing of TNBC and prostate cancer cells were greatly enhanced. This review highlights the potential that using monoclonal antibodies to block LLT1 - NKRP1A interactions could be an effective immunotherapeutic approach to treat triple negative breast cancer and prostate cancer.Item Mechanistic studies of cytotoxic activity of the mesoionic compound MIH 2.4Bl in MCF-7 breast cancer cells(Spandidos Publications, 2020-06-19) Amaral de Mascena Costa, Luciana; Debnath, Dipti; Harmon, Ashlyn C.; de Sousa Araujo, Silvany; Diogenes da Silva Souza, Helivaldo; Filgueiras de Athayde Filho, Petronio; Wischral, Aurea; Adriao Gomes Filho, Manoel; Mathis, J. MichaelIn the present study, the cytotoxic effects of a 1,3-thiazolium-5-thiolate derivative of a mesoionic compound, MIH 2.4Bl, were assessed in the MCF-7 breast cancer cell line. The cytotoxic effects of MIH 2.4Bl were determined using a crystal violet assay. Using a dose-response curve, the IC50 value of MIH 2.4Bl was determined to be 45.8+/-0.8 microM. Additionally, the effects of MIH 2.4Bl on mitochondrial respiration were characterized using oxygen consumption rate analysis. Treating MCF-7 cells with increasing concentrations of MIH 2.4Bl resulted in a significant reduction in all mitochondrial respiratory parameters compared with the control cells, indicative of an overall decrease in mitochondrial membrane potential. The induction of autophagy by MIH 2.4Bl was also examined by measuring changes in the expression of protein markers of autophagy. As shown by western blot analysis, treatment of MCF-7 cells with MIH 2.4Bl resulted in increased protein expression levels of Beclin-1 and ATG5, as well as an increase in the microtubule-associated protein 1A/1B light chain 3B (LC3B)-II to LC3B-I ratio compared with the control cells. Microarray analysis of changes in gene expression following MIH 2.4Bl treatment demonstrated 3,659 genes exhibited a fold-change >/=2. Among these genes, 779 were up-regulated, and 2,880 were down-regulated in cells treated with MIH 2.4Bl compared with the control cells. Based on the identity of the transcripts and fold-change of expression, six genes were selected for verification by reverse transcription-quantitative (RT-q)PCR; activating transcription factor 3, acidic repeat-containing protein, heparin-binding EGF-like growth factor, regulator of G-protein signaling 2, Dickkopf WNT signaling pathway inhibitor 1 and adhesion molecule with Ig like domain 2. The results of RT-qPCR analysis of RNA isolated from control and MIH 2.4Bl treated cells were consistent with the expression changes identified by microarray analysis. Together, these results suggest that MIH 2.4Bl may be a promising candidate for treating breast cancer and warrants further in vitro and in vivo investigation.Item MIEN1 Drives Breast Cancer Invasion by Regulating Cytoskeletal-Focal Adhesions Dynamics(2015-05-01) Kpetemey, Marilyne F.; Vishwanatha, Jamboor K.; Clark, Abbot F.; Basu, AlakanandaIn the recent years, Migration and Invasion Enhancer 1(MIEN1) has emerged as a potential biomarker and a plausible target in breast cancer. Located in the 17q12-21 region of the human chromosome, next to the Her-2/neu loci, MIEN1 presents a robust expression in breast carcinomas; however is completely absent or low in the normal tissues. MIEN1 is post-translationally modified by geranyl-geranyl transferase-I (GgtaseI), which adds isoprenyl group to the carboxyl-terminal of the protein. Prenylated MIEN1 then associates with the inner leaflet of the plasma membrane and acts as an adaptor protein triggering downstream signaling through the Akt/NF-kB axis to regulate the expression of key proteases and angiogenic factors like MMP-9, uPA and VEGF. In migrating cells, MIEN1 enhances filopodium formation at the leading edge. Aside from its prenylation and redox-active motifs, MIEN1 also contains a canonical ITAM, reported to be associated with epithelial-to-mesenchymal transition. Although the role MIEN1 in cell migration and invasion is well known, the underlying molecular mechanisms remain elusive. Here, we show that MIEN1 interacts with Annexin A2, a cytoskeletal protein and a regulator of the plasminogen/plasmin system in breast cancer cells to increase migration and invasion. We confirmed that MIEN1 regulates actin dynamics by associating with cytoskeletal effectors in the lamellum. We also show that MIEN1 expression redirects breast tumor cell migration toward a collective migration. Our studies validate MIEN1-ITAM and CAAX as key motifs to MIEN1-induced functions. In conclusion, our findings confirm the role of MIEN1 in the remodeling of the actin cytoskeleton during motility. Furthermore it attests to previous findings suggesting that motility patterns depend on various environmental factors along with regulatory genes involved. Our study demonstrates an interesting example from cell biology where adaptor proteins regulate various signaling pathways and control cellular processes through protein-protein interactions.Item Nasal Tumor Vaccination Protects against Lung Tumor Development by Induction of Resident Effector and Memory Anti-Tumor Immune Responses(MDPI, 2023-02-26) Donkor, Michael; Choe, Jamie Y.; Reid, Danielle; Quinn, Byron; Pulse, Mark; Ranjan, Amalendu P.; Chaudhary, Pankaj; Jones, Harlan P.Lung metastasis is a leading cause of cancer-related deaths. Here, we show that intranasal delivery of our engineered CpG-coated tumor antigen (Tag)-encapsulated nanoparticles (NPs)-nasal nano-vaccine-significantly reduced lung colonization by intravenous challenge of an extra-pulmonary tumor. Protection against tumor-cell lung colonization was linked to the induction of localized mucosal-associated effector and resident memory T cells as well as increased bronchiolar alveolar lavage-fluid IgA and serum IgG antibody responses. The nasal nano-vaccine-induced T-cell-mediated antitumor mucosal immune response was shown to increase tumor-specific production of IFN-gamma and granzyme B by lung-derived CD8(+) T cells. These findings demonstrate that our engineered nasal nano-vaccine has the potential to be used as a prophylactic approach prior to the seeding of tumors in the lungs, and thereby prevent overt lung metastases from existing extra pulmonary tumors.Item Phosphorlyation of Annexin A2 is essential for its association with exosomes and for migration, invasion and proliferation in triple negative breast cancer(2018-12) Desai, Priyanka P.; Vishwanatha, Jamboor K.; Basha, Riyaz; Chaudhary, PankajExosomes are membrane enclosed small vesicles that range from 40-120 nm in size and participate in cell-cell communication by transferring proteins to other cells. Annexin A2 (AnxA2), a calcium-dependent phospholipid binding protein, is present on the surface of the exosomes. AnxA2 phosphorylation plays an essential role in many physiological conditions by forming a heterotetrameric complex with p11 or S100A10 on the cell surface. We demonstrate here that the phosphorylation at Tyrosine (Tyr)-23 in the N-terminal region of AnxA2 is consequential for its association with the cell surface. This association increases the migratory, invasive and proliferative capacity of MDA-MB-231 triple negative breast cancer (TNBC) cells. An increase in cell surface AnxA2 further leads to a stronger association of AnxA2 with the exosomal surface. We also demonstrate that AnxA2 enriched exosomes promote proliferative and invasive characteristics of a different recipient cell [CAL (Centre Antoine Lacassagne) - 148]. These results demonstrate that Tyr23 phosphorylation of AnxA2 is pivotal for its association with exosomes and for imparting more malignant characteristics to the other breast cancer cells. Thus, AnxA2 could be used as a targeting approach for developing a treatment of TNBC.Item Regulation of Autophagy by Protein Kinase C-epsilon in Breast Cancer Cells(MDPI, 2020-06-15) Basu, AlakanandaProtein kinase C-ɛ (PKCɛ), an anti-apoptotic protein, plays critical roles in breast cancer development and progression. Although autophagy is an important survival mechanism, it is not known if PKCɛ regulates autophagy in breast cancer cells. We have shown that silencing of PKCɛ by siRNA inhibited basal and starvation-induced autophagy in T47D breast cancer cells as determined by the decrease in LC3-II, increase in p62, and decrease in autophagy puncta both in the presence and absence of bafilomycin A1. The mechanistic target of rapamycin (mTOR) associates with Raptor or Rictor to form complex-1 (mTORC1) or complex-2 (mTORC2), respectively. Knockdown of PKCɛ attenuated an increase in autophagy caused by the depletion of Raptor and Rictor. Overexpression of PKCɛ in MCF-7 cells caused activation of mTORC1 and an increase in LC3-I, LC3-II, and p62. The mTORC1 inhibitor rapamycin abolished the increase in LC3-I and p62. Knockdown of mTOR and Rictor or starvation enhanced autophagy in PKCɛ overexpressing cells. While overexpression of PKCɛ in MCF-7 cells inhibited apoptosis, it induced autophagy in response to tumor necrosis factor-ɑ. However, inhibition of autophagy by Atg5 knockdown restored apoptosis in PKCɛ-overexpressing cells. Thus, PKCɛ promotes breast cancer cell survival not only by inhibiting apoptosis but also by inducing autophagy.Item REGULATION OF CELL DEATH BY INHIBITOR OF KAPPA-B KINASE IN TRIPLE NEGATIVE BREAST CANCER CELLS(2013-04-12) Patel, DipaliPurpose: Triple-negative breast cancer (TNBC) is characterized by the lack of estrogen receptor, progesterone receptor, and HER2/neu and hence poses problems for targeted therapy. Thus there is an urgent need to identify suitable molecular targets for the treatment of TNBC. The inhibitor of kappa-B kinase-epsilon (IKK-epsilon) is a breast cancer specific oncogene that is elevated in TNBCs and plays an important role in breast cancer cell survival. Cancer cells often evade death by overcoming apoptosis or type I programmed cell death. However, the role of autophagy or self-digestion in cancer is ambiguous. While a low level of autophagy promotes cell survival under stress, excessive autophagy may lead to type II programmed cell death. The purpose of this project is to determine the mechanism by which IKK-epsilon promotes survival of breast cancer cells. Methods: Triple-negative breast cancer MDA-MB-231 cells were transfected with non-targeting control siRNA or siRNA against appropriate genes. Proteins were separated using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Western blot analysis and enhanced chemiluminescence were used to study the overall levels of apoptosis and autophagy markers. Results: Knockdown of IKK-epsilon in MDA-MB-231 potentiated tumor necrosis factor-alpha (TNF)-induced apoptosis. IKK-epsilon knockdown also showed a decrease in p62/SQSTM1 and an increase in proteins associated with autophagy induction. Conclusions: The increase in autophagic markers in IKK-epsilon depleted cells suggests that IKK-epsilon protects MDA-MB-231 cells by promoting autophagy. Understanding the specific mechanism of survival of TNBC cells could aid in developing effective therapy.