Browsing by Author "Chaudhary, Pankaj"
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Item A Single Site Retrospective Analysis of the Safety and Efficacy of the Amgen COMMUNITY Study(2023-05) Bling, Delaina A.; Chaudhary, PankajIn 2019, a novel respiratory virus surfaced that lead to severe complications in patients and a global pandemic. One of the major issues was COVID-19 associated ARDS. The mortality rate of patients who develop ARDS from a COIVD-19 infection is 45% and at this time there are not many safe and efficacious treatments for the virus. This project exams the Amgen COMMUNITY study at a single site for efficacy and safety. The Amgen study looked at three potential treatments for COVID-19 that would hopefully lessen the occurrence and severity of ARDS; therefore, leading to a lower mortality rate. The efficacy and safety of the Amgen COMMUNITY study was evaluated retrospectively from patients data who participated in the study at the Sunbeam site.Item Analysis of Key Cellular Changes of Triple Negative Breast Cancer Cells in Response to Kinase-Inhibiting BI2536 and Associated Derivatives(2023-05) Baker, Christopher V.; Bunnell, Bruce A.; Burow, Matthew; Chaudhary, PankajPurpose: Triple Negative Breast Cancer (TNBC) is a subtype of breast cancer that grows quickly and has higher rates of metastasis and reoccurrence relative to other Breast Cancer subtypes that make it, in general, a much more dangerous subtype of breast cancer. The Kinase Chemogenomic Set (KCGS) is a collection of 187 kinase-inhibiting compounds with broad activity across 215 different kinases. We hypothesize that this plate contains compounds with the potential to inhibit TNBC and that exploring the transcriptomic and proteomic changes in TNBC cells may give insights into novel treatment targets. Methods: To test this hypothesis, we have utilized two main cell lines, the MDA-MB-231 line and a patient-derived xenograft line, the TuX-BxC-4IC cell line. Measurements have been taken at various time points up until 72 hours at various concentrations of a compound of interest, BI2536, between 1nM and 1uM. Primarily, data will be collected using qRT-PCR to gain insight into the transcriptomic changes during the potential EMT changes. Additionally, various other experiments related to migration, staining, and other essential markers will be conducted on the compound of interest and derivatives of the compound. Results: Initial results and prior work indicate that the compound of interest has moderate success in slowing cancer cell growth. Additionally, initial findings indicate that the compound may succeed in halting and potentially even reversing the EMT process. Conclusion: With this primary data set, we believe that the kinases targeted by the compound may hold potential key targets for the treatment of TNBC.Item Bacteriomimetic Nanoparticles for Immunotherapy against Breast Cancer(2015-03) Kokate, Rutika; Thamake, Sanjay; Chaudhary, Pankaj; Raut, Sangram; Mott, Brittney; Vishwanatha, Jamboor K.Short description: Immunotherapy represents a potential and innovative means to combat cancer. It essentially harnesses the body’s immune system to fight against cancer. Previous literature suggests that 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. In order to surmount this obstacle, several approaches including the use of an “ideal” tumor antigen, appropriate delivery techniques, immune boosting strategies with co-stimulatory molecules are being explored. Purpose: The purpose of this study was to develop “bacteriomimetic nanoparticles” to enhance adaptive cell-mediated immune responses (CD4+ and CD8+ T cell responses) against tumor antigen as a therapeutic option for cancer treatment. Materials and Methods: NPs were prepared by modified solid/oil/water solvent evaporation method using an ultrasonic processor UP200H system (Hielscher Ultrasonics GmbH, Germany). We used membrane preparations of the 4T1 mouse mammary cancer cell line as a tumor antigen and CpG ODN’s as a “bacteriomimetic” stimulant. Fourteen days before tumor challenge BALB/c female mice (6-8 weeks) were pre-immunized with CpG followed by secondary immunization using respective NPs encapsulated with the membrane antigen preparation. Subsequently, mice (n=5) were challenged subcutaneously (SC) with 105 tumor cells and the primary tumor size was monitored using vernier caliper and bioluminiscence imaging (Caliper Life Sciences Inc., MA, USA). Mice were sacrificed on day fourteen after tumor challenge; spleen cells were used for flow cytometric analysis and primary tumor tissue was used to evaluate effect of NP immunization on tumor growth, survival as well as the immune response (CD4+ and CD8+ T cell) via immunohistochemistry. Results: We found significant reduction in progression of tumor growth in mice immunized with CpG coated NPs containing tumor antigen (CpG-NP-Tag). Histological analysis confirmed that tumors in CpG-NP-Tag mice were relatively well differentiated and of lower grade in contrast to CpG-Blank tumors. Immunofluorescence (IF) data further revealed that CpG-NP-Tag tumors had lesser proliferation and higher apoptotic activity. Tumor CD4+ and CD8+T cell infiltration as well as T cell response in spleen was found be higher in CpG-NP-Tag NP immunized mice as compared to the controls (CpG-NP-Blank and NP-Tag). Conclusions: Primary tumor size, IHC, IF and flow cytometry analysis indicate that CpG-NP-Tag NPs were successfully employed to boost the immune response against tumor cells.Item Blocking LLT1-CD161 interaction enhances natural killer cell-mediated lysis of triple-negative breast cancer cells(2018-03-14) Mathew, Stephen O.; Chaudhary, Pankaj; Mathew, Porunelloor A.; Marrufo, Armando M.Purpose: Triple-negative breast cancer (TNBC) accounts for 20 percent of all breast cancer cases and is known to be the most invasive form of breast cancer. TNBC’s absence of estrogen, progesterone, and human epidermal growth factor-2 receptors makes utilizing hormonal treatments ineffective in suppressing tumor growth. TNBC is associated with poorer prognosis and higher incidences of relapse. Therefore, natural killer cell-mediated immunotherapy shows potential as a treatment option for TNBC. Natural killer cells (NK) are innate lymphoid cells that serves its role in the immune system to eradicate infected and tumor cells. NK cell function is regulated through its receptors interacting with activating and inhibitory ligands on target cells. Lectin-like Transcript-1 (LLT1, CLEC2D) is a counter-receptor that interacts with CD161 (NKRP1A) and inhibits NK cell activation. Our study demonstrated that by blocking TNBC’s LLT1 interaction with CD161 with antibodies increases lysis of TNBCs by NK cells. Methods: We have identified the expression and function of LLT1 on TNBC cell lines MDA-MB-231 and MDA-MB-436 by flow cytometry, western blot, immunofluorescent microscopy, and chromium-release assay. LLT1 expression at the cell surface was decreased through gene knockdown with small interference RNA (siRNA) transfection. Primary NK cells were isolated from peripheral blood mononuclear cells from healthy individuals and then were co-incubated with chromium-labeled TNBCs for quantification of specific lysis of TNBCs by NK cells. Results: Our results have demonstrated a higher expression of LLT1 on TNBCs than non-tumorigenic breast cell line MCF10A. We have shown that blocking LLT1 interaction with CD161 with antibodies on TNBCs have increased lysis of TNBCs by primary NK cells. We have also shown that gene knockdown of LLT1 decreases cell surface expression of LLT1 on TNBCs and increases lysis of TNBCs by NK cells. Conclusions: LLT1 expressed on TNBCs is a ligand that interacts with NK receptor CD161 and sends an inhibitory signal to the NK cell thus serving its role for TNBCs to evade immunosurveillance. Respectively, blocking LLT1 with antibodies on TNBCs and decreasing expression of LLT1 by gene knockdown increases susceptibility of TNBCs to NK cell-mediated lysis. Blocking interaction between LLT1 and CD161 with antibodies activates lysis by NK cells and will open a possible new immunotherapeutic strategy for patients diagnosed with TNBC.Item Cell Ghost Coated Polymeric Nanoformulation For Brain Metastasized Triple Negative Breast Cancer(2017-03-14) Van Treuren, Timothy; Ranjan, Amalendu; Chaudhary, Pankaj; Vishwanatha, Jamboor; Kumar, PiyushBackground: Nanoparticle-mediated targeted delivery has become the buzzword in cancer therapy due to its small size and ease in modulation. Cell ghost derived from cancer cells that mimic the cellular environment can be used for specific targeting of tumor in personalized therapy. Purpose: The purpose of this study is to design drug delivery system for personalized medicine to check the brain metastasized Triple Negative Breast Cancer (TNBC). We speculate that cell ghost derived from the tumor of the cancer patient can also be used as a personalized treatment of metastatic cancer. Methods: Cell ghost isolated from brain metastasized TNBC cells was coated on polymeric nanoparticles. Dynamic Light Scattering & Zeta Size analyzer (Malvern Instruments, USA) were used to determine the hydrodynamic size & surface charge of the cell ghost coated nanoparticles(CGNP) respectively. SDS-PAGE was used for comparative analysis of proteins in the cell ghost & CGNP. The Cell uptake of the dye-loaded CGNP was studied using the confocal microscope (Zeiss microscope USA). Results: We have successfully formulated cell ghost coated polymeric nanoparticles (CGNP).The size & surface charge of the CGNP are in desirable range to cross the blood-brain barrier to target brain metastasized TNBC. The SDS-PAGE analysis confirmed that protein contents of cell ghost are stable in CGNP. Confocal microscopic image analysis showed that maximum cellular uptake of these nanoparticles by TNBC cell line. Conclusions: In summary, we concluded that cell ghost isolated from TNBC cells could be used as targeting agents for brain metastatic TNBC. These nanoparticles have maximum cellular uptake and retain the protein contents of cell ghost on nanoformulation infers its possible role in generating personalized medicine for the brain metastasized TNBC treatment.Item Clinical significance of Annexin A2 overexpression in kidney renal clear cell carcinoma(2023) Joseph, Matthew; Chaudhary, PankajPurpose: Invasion and metastasis led to poor prognosis and death of kidney renal clear cell carcinoma (KIRC) patients. In this study, we focus on the characterization of Annexin A2 (AnxA2), which plays an essential role in cell growth, angiogenesis, migration, invasion, and metastasis. Although the role of Annexin A2 (AnxA2) has been studied in many cancers, its function in KIRC is still unexplored. Therefore, in this study, we investigated the AnxA2 expression in tumor tissues of KIRC patients to determine its association with disease characteristics. Methods: We utilized data from The Cancer Genome Atlas (TCGA) to observe AnxA2 gene expression in KIRC and its association with survival. Additionally, immunohistochemical (IHC) analysis was performed to examine the AnxA2 expression in tumor tissues of KIRC patients. Results: In our analysis of TCGA data, AnxA2 mRNA expression was found significantly higher in KIRC tumor tissues compared to the adjacent noncancerous tissues. In addition, AnxA2 expression was significantly associated with higher tumor stage and grade. The high expression of AnxA2 in KIRC patients was significantly correlated to decreased survival [hazard ratio (HR), 1.75; 95% confidence interval (CI), 1.29 - 2.36; p = 0.00023] as compared to low expression. In addition, our IHC staining suggests that AnxA2 was overexpressed in tumor tissues of KIRC patients compared with adjacent noncancerous tissues. Conclusion: AnxA2 is overexpressed in KIRC tumor tissues, and has a direct relationship to the advanced clinicopathological variables and adverse prognosis associated to patients with KIRC.Item Continuous quality improvement at the clinical research site: implementing methods for coordinators in the Heart and Lung Transplant and Pulmonary department at Baylor Scott and White Research Institute(2020-05) Norgan Radler, Charlene R.; Mathew, Stephen O.; Chaudhary, Pankaj; Martinez, Horacio; Felius, JoostNorgan Radler, Charlene R. Continuous quality improvement at the clinical research site: implementing methods for coordinators in the Heart and Lung Transplant and Pulmonary department at Baylor Scott and White Research Institute Master of Science (Clinical Research Management), April 2020 Introduction: The following research project is a Quality Improvement (QI) study to assess resource utilization for six ongoing clinical trials and evaluate the impact of quality improvement methods on the completion of critical trial activities in the Heart and Lung Transplant and Pulmonary (HLTP) department at Baylor Scott and White Research Institute (BSWRI). Methods: The project design is a case series in which observations were made on research staff before and after an intervention, with no control group. Non-probability sampling with purposeful, maximum variation was used due to the study's qualitative research design. Metrics were collected regarding the completion of key trial activities of subject screening, subject enrollment, and data entry before and after intervention using a spreadsheet tool. Collected metrics were reviewed to identify areas for improvement and QI interventions were designed and implemented to reallocate resources as appropriate. The data was maintained in a run chart to monitor changes during the pre-intervention and post-intervention periods. Statistical analysis was performed on the data to evaluate the effect of the intervention. Results: The Wilcoxon Signed-Rank test was used to statistically analyze differences in medians of activity metrics across all studies before and after intervention. All variables improved in the direction of the applied interventions except time screening subjects and data entered in the electronic data capture (EDC) system. Median differences were found statistically non-significant, except the combined variable of number of open queries and case report forms (CRF) not entered weekly which demonstrated a statistically significant decrease following intervention. Median time screening subjects demonstrated a non-significant decrease following intervention while median number of subjects screened showed a non-significant increase. Median time enrolling subjects and median number of subjects enrolled increased post intervention, but statistical testing was not performed due to the small sample size below the minimum critical threshold required. Median time entering data in the EDC demonstrated a non-significant increase following intervention while median number of CRFs entered in the EDC showed a non-significant decrease. Conclusion: Implementation of the quality improvement process for clinical research staff provided a tool for our site to continuously assess and improve trial outcomes. Five of the seven variables receiving quality improvement interventions improved in the direction of the intervention, with one demonstrating a statistically significant difference. The small sample size used may have decreased the power of the study to detect statistical significance. Future studies should be completed to apply the quality improvement methodology used to a larger sample size. In conclusion, this study established 'proof of concept' for the completion of future, larger-scale quality improvement projects at our research site.Item Correction to: Serum exosomal-annexin A2 is associated with African-American triple-negative breast cancer and promotes angiogenesis(BioMed Central Ltd., 2020-03-23) Chaudhary, Pankaj; Gibbs, Lee D.; Maji, Sayantan; Lewis, Cheryl M.; Suzuki, Sumihiro; Vishwanatha, Jamboor K.After publication of the original article [1], we were notified that the wrong version of Fig. 2b has been published.Item Effects of Ionizing Radiation on Human Triple Negative Breast Cancer(2020) Joshi, Rohan; Chaudhary, Pankaj; Jones, Harlan; Choe, Jamie Y.Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype which exhibits high rates of metastasis. Due to lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor-2 receptor (HER2), TNBC is not responsive to hormonal treatment and currently lacks targeted therapies. Radiation is an important component of cancer therapy and used clinically in combination with chemotherapy and/or surgery for TNBC patients. Specialized extracellular vesicles called exosomes are involved in intercellular communication and postulated to play roles in tumor metastasis. In this study, we evaluate effects of single-dose ionizing radiation on the proliferation and release of exosomes from three human TNBC cell lines (MDA-MB-231, CAL-51, HCC-1806). TNBC cells were cultured and irradiated with 8.6 Gy. Exosomes were isolated 72h post-irradiation using differential ultracentrifugation and evaluated for size and purity. Nanoparticle tracking analysis (NTA) quantified exosomes released by tumor cells. Western blot confirmed isolation of exosomes by determining expression of established exosome membrane protein markers. Ionizing radiation suppressed proliferation and migration of MDA-MB-231, CAL-51, and HCC-1806 based on in vitro wound-healing (scratch) assays. Total cell counts for irradiated vs. control conditions at the time of exosome isolation were statistically significant in CAL-51 (P=0.0205) and HCC-1806 (P=0.0261). Exosomes released per cell in response to radiation showed inconclusive trends. Our preliminary data demonstrates radiation is capable of depressing TNBC proliferation and warrants further exploration regarding radiation-induced effects on tumor-derived exosomes.Item Enhancing the Effectiveness of Serious Adverse Event Reporting in Clinical Trials: A Framework for Continuous Improvement(2023-12) Bonsu, Chelsea O.; Chaudhary, PankajItem HIGH EXPRESSION OF MIEN1 IN BREAST CANCER CORRELATES WITH POOR SURVIVAL OUTCOME(2019-03-05) Chaudhary, Pankaj; Vishwanatha, Jamboor; Nsiah, Nana YaaPURPOSE: Breast cancer is the second leading cause of cancer-related deaths in women. Metastasis accounts for majority of breast cancer deaths. It remains a major barrier to cancer treatment due to limitations in diagnosis and lack of effective therapy. Understanding the role of underlying molecular mechanisms involved in metastasis could lead to effective therapy to prevent and treat breast cancer. Migration and Invasion Enhancer 1 (MIEN1), is a novel gene abundantly expressed in different tumors compared to normal cells. Our previous studies have shown it plays a critical role in regulating cell migration and invasion to promote metastases. It is located on chromosome 17q12 near the Her 2/neu oncogene. MIEN1 protein is a membrane-anchored signal protein, with important structural motifs such as the Immunoreceptor tyrosine-based activation motif (ITAM), a redox active motif and a prenylation sequence at the carboxyl terminal. Here, we evaluated the expression of MIEN1 in breast cancer patients with clinical outcome. METHODS: We analyzed The Cancer Genome Atlas (TCGA) Breast Invasive Carcinoma (BRCA) database to observe MIEN1 mRNA expression in breast cancer subtypes and its correlation with survival. Also, we assessed MIEN1 expression in a panel of normal and breast cancer cell lines using Western blot. RESULTS: MIEN1 gene expression was significantly increased in different subtypes of breast carcinomas (Invasive ductal carcinoma, Invasive lobular carcinoma, Mixed Ductal and Lobular, and Mucinous) compared to normal tissues. Moreover, MIEN1 is predominantly overexpressed in HER2+ breast cancer patients compared to other subtypes. However, MIEN1 expression in luminal A, luminal B and basal-like subtypes were also high in comparison to normal breast tissues. High expression levels of MIEN1 was associated with reduced overall survival (HR = 1.61; 95% CI = 1.34-1.94, P = 0.0001). Screening of MIEN1 expression in various breast cancer cell lines suggest that expression of MIEN1 is high in majority of them compared to immortalized normal mammary epithelial cell line. CONCLUSION: Our findings confirm that MIEN1 is an important oncogene, and its increased expression in breast cancer contributes towards an aggressive disease with poor survival.Item High expression of N-acetyl transferase 9 in cholangiocarcinoma and its possible role in tumor progression(2024-03-21) Hoteit, Tamara; Jones, Harlan; Chaudhary, PankajCholangiocarcinoma, CCA, is an aggressive type of liver cancer due to the scarce number of biomarkers and its resistance to anticancer drugs, leading to difficulty in its early detection. Its 5-year survival rate ranges from 2% to 24%, depending on severity and metastasis. Currently, surgery is the most effective treatment option, but only under certain criteria, such as the cancer being caught early and having not metastasized. CCA incidence is the highest in Asian countries because of the presence of a carcinogenic liver fluke. N-acetyl transferase, NAT, is an enzyme with many functions, including regulating protein stability, membrane targeting, gene silencing, and drug resistance. Different subsets of NATs are also known to be biomarkers for different types of cancer, such as colorectal, prostate, and breast cancers. According to The Cancer Genome Atlas database, NAT9, a subset of the NAT family, is overexpressed in patients with CCA; however, no studies have demonstrated its role in CCA, indicating the need for more research. In this study, we aim to assess the expression of NAT9 in CCA using cell lines and patient tissue samples through RT-qPCR, western blotting, and immunohistochemistry. This data will help us shed light on NAT9’s role in the tumorigenesis of CCA and could be a promising biomarker and therapeutic target for CCA.Item Higher expression of Annexin A2 in bladder urothelial carcinoma promotes migration and invasion.(2023) Guo, Christina; Chaudhary, PankajPurpose:Bladder cancer is a prevalent and often aggressive malignancy, precipitating high morbidity and mortality rates in the US. The paucity of non-invasive and low-cost methods for detection necessitates investigation into improved surveillance assays for bladder urothelial carcinoma (BLCA). Annexin A2 (AnxA2) is a phospholipid-binding protein involved in malignant processes in several cancers but has yet to be studied in association with bladder carcinoma. This study aims to investigate the association of AnxA2 in BLCA and establish its role in the metastasis of bladder cancer cells. Results:The Cancer Genome Atlas Data analysis demonstrated that AnxA2 mRNA expression was significantly increased in BLCA tumor tissue compared to normal bladder tissue. Higher AnxA2 mRNA expression was significantly associated with high pathological grades, stages, and non-papillary tumor histology and poor overall survival, progression-free survival, and disease-specific survival. Similarly, we found that AnxA2 expression was higher in bladder cancer cells derived from high-grade metastatic carcinoma than in cells derived from low-grade urothelial carcinoma. In addition, expression of AnxA2 was significantly mobilized to the surface of bladder cancer cells that were highly metastatic versus cancer cells derived from low-grade tumors. This expression was also associated with high plasmin generation and AnxA2 secretion. Downregulation of AnxA2 cells significantly inhibited proliferation, migration, and invasion in bladder cancer and decreased expression of proangiogenic growth factors and cytokines. Conclusion: Results of this study show that higher expression of AnxA2 is involved in proliferation, migration, and invasion in bladder cancer and is associated with poor prognosis of patients with BLCA. These findings demonstrate the potential for AnxA2 as a prognostic marker and therapeutic target for BLCA. Funding: This research was partly supported by the NIH grants CA233355 and HL125447. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.Item Higher Expression of Annexin A2 in Metastatic Bladder Urothelial Carcinoma Promotes Migration and Invasion(MDPI, 2022-11-27) Guo, Christina; Trivedi, Rucha; Tripathi, Amit K.; Nandy, Rajesh; Wagner, Diana C.; Narra, Kalyani; Chaudhary, PankajIn this study, we aim to evaluate the significance of AnxA2 in BLCA and establish its metastatic role in bladder cancer cells. Analysis of TCGA data showed that AnxA2 mRNA expression was significantly higher in BLCA tumors than in normal bladder tissues. High mRNA expression of AnxA2 in BLCA was significantly associated with high pathological grades and stages, non-papillary tumor histology, and poor overall survival (OS), progression-free survival (PFS), and diseases specific survival (DSS). Similarly, we found that AnxA2 expression was higher in bladder cancer cells derived from high-grade metastatic carcinoma than in cells derived from low-grade urothelial carcinoma. AnxA2 expression significantly mobilized to the surface of highly metastatic bladder cancer cells compared to cells derived from low-grade tumors and associated with high plasmin generation and AnxA2 secretion. In addition, the downregulation of AnxA2 cells significantly inhibited the proliferation, migration, and invasion in bladder cancer along with the reduction in proangiogenic factors and cytokines such as PDGF-BB, ANGPT1, ANGPT2, Tie-2, bFGF, GRO, IL-6, IL-8, and MMP-9. These findings suggest that AnxA2 could be a promising biomarker and therapeutic target for high-grade BLCA.Item Impact of Annexin A2 on virus life cycles(Elsevier B.V., 2024-05-04) Park, In-Woo; Fiadjoe, Hope K.; Chaudhary, PankajDue to the limited size of viral genomes, hijacking host machinery by the viruses taking place throughout the virus life cycle is inevitable for the survival and proliferation of the virus in the infected hosts. Recent reports indicated that Annexin A2 (AnxA2), a calcium- and lipid-binding cellular protein, plays an important role as a critical regulator in various steps of the virus life cycle. The multifarious AnxA2 functions in cells, such as adhesion, adsorption, endocytosis, exocytosis, cell proliferation and division, inflammation, cancer metastasis, angiogenesis, etc., are intimately related to the various clinical courses of viral infection. Ubiquitous expression of AnxA2 across multiple cell types indicates the broad range of susceptibility of diverse species of the virus to induce disparate viral disease in various tissues, and intracellular expression of AnxA2 in the cytoplasmic membrane, cytosol, and nucleus suggests the involvement of AnxA2 in the regulation of the different stages of various virus life cycles within host cells. However, it is yet unclear as to the molecular processes on how AnxA2 and the infected virus interplay to regulate virus life cycles and thereby the virus-associated disease courses, and hence elucidation of the molecular mechanisms on AnxA2-mediated virus life cycle will provide essential clues to develop therapeutics deterring viral disease.Item INHIBITION OF TRIPLE NEGATIVE AND HER-2 RESISTANT BREAST CANCER PROGRESSION BY ANNEXIN A2 ANTIBODIES(2013-04-12) Chaudhary, PankajPurpose: Herceptin, an immunotherapy directed against the Her-2 receptor, inhibits cancer growth and progression in Her-2 positive breast cancer by blocking the downstream survival pathways. In these cells however, the expression of another major tyrosine kinase receptor EGFR is significantly low. Interestingly, EGFR expression is significantly increased in Her-2 negative breast cancer thereby contributing to cancer growth and progression. Present studies were designed to investigate whether or not Annexin A2 (AnxA2), a calcium dependent phospholipid binding protein, regulated EGFR downstream signaling pathway and if the functions of EGFR can be inhibited by the AnxA2 antibody in TNBC and Herceptin resistant cancer cell lines. Methods: Triple negative breast cancer cell line MDA-MB-231 and Her-2 resistant breast cancer cell line JIMT-1 were grown in complete DMEM and DMEM/F12 medium respectively, in a humidified incubator at 37C with 5% CO2. The AnxA2 function at cell surface was blocked incubating with AnxA2 antibody (2µg/ml) after 12 h of serum starvation. The cells were treated with/without EGF (50ng/ml) for 20 min after 2 h of antibody treatment. The cell lysate was analyzed for pEGFR and EGFR mediated downstream signaling by Western blotting. Results: The results of the present study indicate that AnxA2 interacts with EGFR at the cell surface and plays an important role in the regulation of EGFR mediated downstream signaling. Treatment of MDA-MB-231 and JIMT-1 cells with AnxA2 antibody causes significant decrease in EGF-mediated phosphorylation of EGFR at Y845 and Y1068 sites. In addition, treatment of cells with AnxA2 antibody decreases the ligand induced EGFR dimerization and internalization. Our results also demonstrate that blocking cell surface AnxA2 functions causes the downregulation of proteins such as pAKT, and pERK1/2 which are regulated by EGFR resulting in lower cell survival, proliferation, and migration. Conclusions: These studies indicate that association of AnxA2 with EGFR in the membrane domain might play a positive regulatory role in keeping EGFR signaling events in an activated state in triple negative and Her-2 resistant breast cancer thus making AnxA2 an important therapeutic target.Item LLT1-Mediated Immunotherapy for Hepatocellular Carcinoma(2022-08) Allison, Michaela M.; Mathew, Stephen O.; Chaudhary, Pankaj; Mathew, Porunelloor A.; Forster, Michael J.Item MIEN1 Regulates Breast Cancer Cell Migration and Invasion by Altering Cytoskeletal Dynamics Through Focal Adhesion Kinase and N-WASP Signaling(2018-03-14) Chaudhary, Pankaj; Kpetemey, Marilyne; Vishwanatha, Jamboor; Van Treuren, TimothyPurpose: Triple negative breast cancer (TNBC), accounts for approximately 15-20% of all breast cancer diagnoses. This is the most aggressive breast cancer subtype and is characterized by a lack of known receptors associated with, making prognosis and treatment difficult in patients with TNBC. TNBC has a propensity to metastasize to vital organs, including lung, brain and bone. This can occur early in the disease progression and usually leads to the elevated mortality rate in TNBC patients. Research efforts to identify molecular markers within TNBC for prognosis and therapy have not been fruitful. Migration and Invasion Enhancer 1 (MIEN1) has been implicated in the disease progression of many cancers, including TNBC. We determined to further understand the molecular mechanisms by which MIEN1 regulates cell motility and invasion in the context of TNBC. This knowledge will provide a basis to pursue MIEN1 as a potential marker for future treatment and evaluation of TNBC cases. Methods: Wild-type MIEN1 (MIEN1-WT) or Immunoreceptor tyrosine-based activation motif (ITAM)-mutant MIEN1 MIEN1-Y39/50F) was overexpressed in MDA-MB-231 cells to evaluate the role of ITAM signaling in MIEN1 mediated migration and invasion. Migration speed and persistence toward a chemoattractant was assessed using microfluidic chambers. Invasion was evaluated by embedding cell aggregates in a 3D collagen matrix and examining the spread of the cells. MIEN1 influence on migration was mediated by actin cytoskeletal dynamics. This mechanism was further delineated by looking at actin polymerization as well as focal adhesion adaptors and signaling molecules using western blotting as well as confocal microscopy. An in vitro kinase assay was also used to evaluate activators of MIEN1. Results: MIEN1-WT over-expression in MDA-MB-231 cells resulted increased migratory and invasive capabilities compared to wild-type cells. Additionally, over-expression of the MIEN1-Y39/50F ITAM mutant inhibited the cells’ ability to migrate towards a chemoattractant as well as invade through a collagen matrix. MIEN1 co-localized to the cell membrane with FAK (focal adhesion kinase) and facilitated signaling through N-WASP to alter cytoskeletal dynamics and increase filamentous actin accumulation. Conclusion: MIEN1 regulates migration and invasion of TNBC cells by altering cytoskeletal dynamics through activation of FAK and N-WASP, which results in increased actin polymerization and cell motility.Item MIMICKING INFECTION FOR IMMUNOTHERAPY AGAINST BREAST CANCER-FOOLING THE IMMUNE SYSTEM(2014-03) Kokate, Rutika; Thamake, Sanjay; Chaudhary, Pankaj; Mott, Brittney; Vishwanatha, Jamboor K.; Jones, Harlan P.Immunotherapy represents a potential and innovative means to combat cancer. It essentially harnesses the body’s immune system to fight against cancer. Previous literature suggests that 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. In order to surmount this obstacle, several approaches including the use of an “ideal” tumor antigen, appropriate delivery techniques, immune boosting strategies with co-stimulatory molecules are being explored. Purpose (a): The purpose of this study was to develop “bacteriomimetic nanoparticles” to enhance adaptive cell-mediated immune responses (CD4+ and CD8+ T cell responses) against tumor antigen as a therapeutic option for cancer treatment. Methods (b): NPs were prepared by modified solid/oil/water solvent evaporation method using an ultrasonic processor UP200H system (Hielscher Ultrasonics GmbH, Germany). We used membrane preparations of the 4T1 mouse mammary cancer cell line as a tumor antigen and CpG ODN’s as a “bacteriomimetic” stimulant. Fourteen days before tumor challenge BALB/c female mice (6-8 weeks) were pre-immunized with CpG followed by secondary immunization using respective NPs encapsulated with the membrane antigen preparation. Subsequently, mice (n=4) were challenged with 105 tumor cells intravenously (IV). Mice were sacrificed and tumors were harvested at days 3, 7 and 14 respectively. CD4+ and CD8+ T cell responses were measured in lower respiratory node and spleen using flow cytometry. In another experimental set, following the same immunization schedule as mentioned above, mice (n=5) were challenged subcutaneously (SC) with 105 tumor cells. Primary tumor size was monitored using vernier caliper and bioluminiscence imaging (Caliper Life Sciences Inc., MA, USA). Mice were sacrificed on day fourteen after tumor challenge; spleen cells were used for flow cytometric analysis and primary tumor tissue was used to evaluate CD4+ and CD8+ T cell via immunohistochemistry. Results (c): We found significant reduction in progression of tumor growth in mice immunized with CpG coated NPs containing tumor antigen (CpG-NP-Tag). Histological analysis confirmed that tumors in CpG-NP-Tag mice were relatively well differentiated and of lower grade in contrast to CpG-Blank tumors. Immunofluorescence (IF) data further revealed that CpG-NP-Tag tumors had lesser proliferation and higher apoptotic activity. Tumor CD4+ T cell infiltration as well as T cell response in spleen was found be higher in CpG-NP-Tag NP immunized mice as compared to the controls. Conclusions (d): Primary tumor size, IHC, IF and flow cytometry analysis indicate that CpG-NP-Tag NPs were successfully employed to boost the immune response against tumor cells.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.