Search

Effect of ORM on cell cycle analysis and β-catenin localization. A. Histogram (i) and table (ii) represent the cell cycle distribution in DU145 cells. B. Confocal images of β-catenin, DAPI and merged images of control (i) and ORM treated (ii) PC3 cells.

Ormeloxifene is a clinically approved selective estrogen receptor modulator, which has also shown excellent anticancer activity, thus it can be an ideal repurposing pharmacophore. Herein, we report therapeutic effects of ormeloxifene on prostate cancer and elucidate a novel molecular mechanism of its anticancer activity. Ormeloxifene treatment inhibited epithelial-to-mesenchymal transition (EMT) process as evident by repression of N-cadherin, Slug, Snail, vimentin, MMPs (MMP2 and MMP3), β-catenin/TCF-4 transcriptional activity, and induced the expression of pGSK3β. In molecular docking analysis, ormeloxifene showed proficient docking with β-catenin and GSK3β. In addition, ormeloxifene induced apoptosis, inhibited growth and metastatic potential of prostate cancer cells and arrested cell cycle in G0–G1 phase via modulation of cell-cycle regulatory proteins (inhibition of Mcl-1, cyclin D1, and CDK4 and induction of p21 and p27). In functional assays, ormeloxifene remarkably reduced tumorigenic, migratory, and invasive potential of prostate cancer cells. In addition, ormeloxifene treatment significantly (P < 0.01) regressed the prostate tumor growth in the xenograft mouse model while administered through intraperitoneal route (250 μg/mouse, three times a week). These molecular effects of ormeloxifene were also observed in excised tumor tissues as shown by immunohistochemistry analysis. Our results, for the first time, demonstrate repurposing potential of ormeloxifene as an anticancer drug for the treatment of advanced stage metastatic prostate cancer through a novel molecular mechanism involving β-catenin and EMT pathway. Mol Cancer Ther; 16(10); 2267–80. ©2017 AACR.

Supplementary information. Supplementary methods and additional results as referred to in the manuscript

Supplementary File 3. Sixty-three core genes from the mouse metastatic model that are enriched in the developmental co-expression module

Supplementary File 5. Promoter regions from MURINE NFATc1 target genes showing the locations of NFAT consensus binding sequences and PCR primers.

Supplementary Figures 1-9. All supplementary data for publication as referenced in the manuscript

Data Supplement from Nuclear Factor of Activated T-cell Activity Is Associated with Metastatic Capacity in Colon Cancer

Supplementary Tables. All tables containing supplementary data for publication as referenced in the manuscript

Supplmentary File 2. Genes in 441 co-expression modules identified within the integrated colon cancer co-expression network.

Supplementary File 4. Promoter regions from HUMAN NFAT family target genes shown in Figure 1E, showing location of NFAT consensus binding sequences

Malaria is among the top-ranked parasitic diseases that pose a threat to the existence of the human race. This study evaluated the antimalarial effect of the rhizome of Zingiber officinale in infected mice, performed secondary metabolite profiling and detailed computational antimalarial evaluation through molecular docking, molecular dynamics (MD) simulation and density functional theory methods. The antimalarial potential of Z. officinale was performed using the in vivo chemosuppressive model; secondary metabolite profiling was carried out using liquid chromatography–mass spectrometry (LC–MS). Molecular docking was performed with Autodock Vina while the MD simulation was performed with Schrodinger desmond suite for 100 ns and DFT calculations with B3LYP (6-31G) basis set. The extract showed 64% parasitaemia suppression, with a dose-dependent increase in activity up to 200 mg/kg. The chemical profiling of the extract tentatively identified eight phytochemicals. The molecular docking studies with plasmepsin II and Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) identified gingerenone A as the hit molecule, and MMGBSA values corroborate the binding energies obtained. The electronic parameters of gingerenone A revealed its significant antimalarial potential. The antimalarial activity elicited by the extract of Z. officinale and the bioactive chemical constituent supports its usage in ethnomedicine. Communicated by Ramaswamy H. Sarma

Colorectal cancer (CRC) is the second deadliest cancer, and over 40-50% of patients develop metastasis during their fight against the disease. CRC survival rate drops from 90% to 14% when the condition is contained within the colon vs. when found at distant sites within the body. Metastasis is a multistep process and one of the critical steps for cancer cells to acquire anoikis resistance to survive after detachment from the primary sites and travel through the circulatory and lymphatic systems to distant target organs. Thus, understanding the molecular players involved in the anoikis process and metastasis could be vital for improving the survival of CRC patients. The aberrant expression of a long noncoding RNA (lncRNA) urothelial carcinoma-associated 1 (UCA1) has been identified in CRC. However, its role in metastasis processes is not yet well defined. Our preliminary results in the anchorage-independent growth (anoikis model) demonstrate increased expression of lncRNA UCA1. Moreover, the overexpression of lncRNA UCA1 led to high expression of stemness markers SOX2 and Kaiso, along with increased survival of anchorage-independent cells, which indicates a potential mechanistic role of UCA1 in anoikis resistance. Thus, in this study, we propose elucidating the role(s) of UCA1 and its associated signaling pathways during anoikis resistance. We hypothesize that the overexpression of lncRNA UCA1 enhances CRC metastasis through anoikis resistance-associated signaling pathways. We will utilize Isogenic CRC cell lines SW480 (oncogenic) and SW620 (metastatic) to understand the mechanistic regulation of anoikis resistance. Lentiviral transduced stable overexpression (SW480+UCA1//GFP) and knockdown (SW620+CRISPRgUCA1) cell lines have been utilized for this study. After subjecting these cell lines (along with control) to anchorage-independent growth conditions, cell cycle, pro-survival, anti-apoptotic, stemness, and glucose metabolism factors will be analyzed through RT-PCR, Digital Droplet PCR (ddPCR), western blot, and FACS analyses. Utilizing the same model, we will examine lncRNA UCA1 linked anoikis resistance specific phosphorylation profiles of kinases, their protein substrates using the Proteome Profiler Phospho-Human Phospho-Kinase Array. Citation Format: Sophia Leslie, Kyle D. Doxtater, Samantha Lopez, Kristopher Ezell, Adithya Anilkumar, Amayrani Sanchez, Bilal Hafeez, Tamer Oraby, Meena Jaggi, Timothy Loy, Subhash Chauhan, Manish K. Tripathi. Signaling pathways modulated by lncRNA UCA1 during anchorage-independent growth in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1291.

The Y-Box Binding protein 1 (YB-1) is a 324 amino acid-long member of the Cold Shock Domain (CSD) protein superfamily. YB-1 interacts with micro RNAs and non-coding RNAs in the cytoplasm, acting as a regulator of mRNA translation. YB-1 translocation into the nucleus is circadian clock-dependent and leads to the upregulation of cyclin gene expression, among others. Another mechanism of nuclear translocation of YB-1 is mediated by phosphorylation of the Ser 102 residue in the CSD. The YB-1 oncoprotein acts as a transcription factor when bound to DNA, modulating the transcription of genes that regulate biological processes such as Multidrug Resistance Mutation 1 (MDR1). YB-1 is linked to poor prognosis in breast, prostate, and liver cancer. Despite advances in biomedicine, the incidence and mortality of hepatocellular carcinoma (HCC) remain high. Sorafenib, a tyrosine kinase Inhibitor (TKI), is the first-line treatment for advanced HCC. Sorafenib also inhibits downstream activation of NF-kB by inhibition of Lyn phosphorylation (Y397). However, it was observed in many patients that its sorafenib’s effectiveness is hampered by drug resistance, but the mechanism is still unclear. We have found that SK-HEP-1 HCC cells transfected with a YB-X-1 overexpression plasmid subsequently developed into a stable YB-1 overexpression cell line (SK-HEP-1+YB-1) displayed increased invasion, migration, proliferation, and colony formation property as compared to the vector control (SKHEP1+Vec). The SK-HEP-1+YB-1 cell line also showed increased IC50 for sorafenib and elevated phosphorylation of Lyn (Y397) and further activated NF-kB (S165/S176) phosphorylation. This indicates YB-1 may cause increased Sorafenib resistance in HCC by activating tyrosine kinases (TK), such as lyn, and simultaneous activation of NF-kB. The combination of YB-1 inhibition and sorafenib might provide a novel therapeutic strategy and improve the survival of patients with advanced-stage HCC. Citation Format: Ana G. Ayala Pazzi, Omar Karkoutly, Kristopher Ezell, Samantha Lopez, Kyle D. Doxtater, Elias George, Vijian Dhevan, Manish K. Tripathi. Oncoprotein YB-1 modulation of Lyn-MAPK-NF-kB pathway, a potential therapeutic strategy combined with sorafenib for advanced stage HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1281.

Gastrointestinal (GI) cancer affects various significant organs along the gastrointestinal tract, which contains several of the leading cancer-related deaths in the United States. According to the American Cancer Society, colorectal and liver cancers are among the top ten leading deaths due to cancer, ranking third and sixth, respectively. In the Rio Grande Valley region with a majority Hispanic population, colorectal cancer and liver have a higher occurrence than in the entire state of Texas. However, for many, a cancer diagnosis can worsen with stress, which is also a common occurrence within the Rio Grande Valley region due to 30% of its population living in poverty. Stress can cause changes to the immune system's functionality and the inflammatory response that could lead to insufficient surveillance for any potential oncogenicity. When the Hypothalamic-Pituitary-Adrenal (HPA) axis becomes activated by stress, it will release several stress hormones, such as leptin and cortisol, in response that can play a role in the progression and growth of cancer tumors. To further explore this relationship, hepatocellular carcinoma (HCC) cells and colorectal cancer (CRC) cells were treated with cortisol and leptin at various time points to create models mimicking acute and chronic stress exposure. Afterward, the mRNA mean fold change levels were obtained utilizing the RT-PCR method to determine the levels of each gene in various liver and colorectal cancer cell lines.Additionally, a kinase assay will provide further insight and evidence into the signaling pathway based on the results of the RT-PCR. The current results have shown an upregulation of lncRNA MALAT1, ZNF384, and YB-1 genes at varying degrees depending on the cell line, stress hormone, and duration of treatment. Consequently, these findings have shown an upregulation of oncogenic long non-coding RNA and transcription factors, presenting a signaling oncogenic pathway model by stress factors in HCC and CRC cell lines. Therefore, understanding the role of stress-factors in the oncogenicity and progression of cancer can aid in creating therapeutics for a population where stress can play a role in cancer disparity. Citation Format: Amayrani Sanchez, Kyle D. Doxtater, Samantha Lopez, Sophia Leslie, Elias George, Vijian Dhevan, Meena Jaggi, Subhash Chauhan, Manish K. Tripathi. Signaling pathways associated with stress factors in gastrointestinal cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4822.

Cancer metastasis is one of the deadliest aspects of the disease, with about 90% of all cancer-related deaths due to its development at different sites within the body. Colorectal cancer (CRC) is the second leading cause of cancer mortality in the United States, with 40-50% of all patients developing metastasis at some point during their fight with the disease. With the approval of Regorafenib for treating metastatic colorectal cancer, steps have been taken to combat metastasis in colorectal cancer. A vital aspect of the development of metastasis is the development of resistance to first-line chemotherapy. Regorafenib is an oral small-molecule multiple kinase inhibitor. It is indicated worldwide for patients with metastatic colorectal cancer (mCRC), Regorafenib has already begun to show resistance in CRC. Understanding the mechanisms behind Regorafenib resistance in CRC is vital. Studies have demonstrated the expression of Long-non-coding RNA (LncRNA) to be linked to cancer metastasis and drug resistance. LncRNA UCA1 has been shown in other cancers to lead to resistance to different drugs like cisplatin, gemcitabine, 5-FU, tamoxifen, imatinib, and EGFR-TKIs. In our lab, we have found the LncRNA UCA1 to be overexpressed in CRC patient tissues, with increasing expression across stages I-III, compared to normal tissue. High UCA1 expression has decreased survival among colorectal cancer patients, per the TCGA patient cohort analysis. Furthermore, we found that high UCA1 expression in colorectal cancer cell lines leads to high IC50 values for Regorafenib. Lentiviral transduce stable overexpression (SW480), and knockdown (SW620) cell lines were developed for UCA1-regorafenib drug resistance mechanistic studies. Increased expression of UCA1 led to increased expression of crucial drug resistance genes (MDR1, ABCB1, and FOXM1) and increased IC50 compared to the control vector. In contrast, the knockdown of UCA1 led to decreased expression of resistance markers and IC50 in SW620 cells. A 3D spheroid model was utilized to assay regorafenib sensitivity to the UCA1 overexpressed and knockdown cell lines. High UCA1 expression leads to the formation of a higher number of 3D spheroid bodies and size when compared to vector. Furthermore, when treating the spheroid with IC50 concentration, UCA1 overexpressing spheroids showed higher vitality and increased size compared to the vector control. We have also analyzed the signaling pathways modulated by UCA1 in CRC cell lines, which may be involved in enhancing the regorafenib resistance. This supports the notion that UCA1 is critical in enhancing the resistance to regorafenib in CRC by activating drug resistance pathways. For the first time, this study demonstrates that UCA1 provides drug resistance to regorafenib in CRC, facilitating the progression of CRC metastasis. Citation Format: Kyle D. Doxtater, Manish K. Tripathi. LncRNA UCA1 as a potential therapeutic target for drug resistance in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2854.

Pancreatic cancer has the worst prognosis and lowest survival rate among all cancers. Pancreatic cancer cells are highly metabolically active and typically reprogrammed for aberrant glucose metabolism; thus they respond poorly to therapeutic modalities. It is highly imperative to understand mechanisms that are responsible for high glucose metabolism and identify natural/synthetic agents that can repress glucose metabolic machinery in pancreatic cancer cells, to improve the therapeutic outcomes/management of pancreatic cancer patients. We have identified a glycoside, steviol that effectively represses glucose consumption in pancreatic cancer cells via the inhibition of the translation initiation machinery of the molecular components. Herein, we report that steviol effectively inhibits the glucose uptake and lactate production in pancreatic cancer cells (AsPC1 and HPAF-II). The growth, colonization, and invasion characteristics of pancreatic cancer cells were also determined by in vitro functional assay. Steviol treatment also inhibited the tumorigenic and metastatic potential of human pancreatic cancer cells by inducing apoptosis and cell cycle arrest in the G1/M phase. The metabolic shift by steviol was mediated through the repression of the phosphorylation of mTOR and translation initiation proteins (4E-BP1, eIF4e, eIF4B, and eIF4G). Overall, the results of this study suggest that steviol can effectively suppress the glucose metabolism and translation initiation in pancreatic cancer cells to mitigate their aggressiveness. This study might help in the design of newer combination therapeutic strategies for pancreatic cancer treatment.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has disrupted social and economic life globally. The global pandemic COVID-19 caused by this novel SARS-CoV-2 shows variable clinical manifestations, complicated further by cytokine storm, co-infections, and coagulopathy, leading to severe cases and death. Thrombotic complications arise due to complex and unique interplay between coronaviruses and host cells, inflammatory response, and the coagulation system. Heparin and derivatives are World Health Organization (WHO) recommended anticoagulants for moderate and severe Corona Virus Disease 19 (COVID-19), that can also inhibit viral adhesion to the cell membrane by interfering with heparan sulfate-dependent binding to angiotensin-converting enzyme 2 (ACE2) receptor. Heparin also possesses anti-inflammatory, immunomodulatory, antiviral, and anti-complement activity, which offers a benefit in limiting viral and microbial infectivity and anticoagulation from the immune-thrombosis system. Here we present a case study of the pathophysiology of unexpected COVID-19 coagulopathy of an obese African American patient. While being on therapeutic warfarin since admission, he had a dismal outcome due to cardio-pulmonary arrest after the sudden rise in D-dimer value from 1.1 to >20. This indicates that for such patients on chronic warfarin anticoagulation with “moderate COVID 19 syndromes”, warfarin anticoagulation may not be suitable compared to heparin and its derivatives. Further research should be done to understand the beneficial role of heparin and its derivatives compared to warfarin for COVID-19 inflicted patients.

The well-defined and characterized 3D crystal structure of a protein is important to explore the topological and physiological features of the protein. The distinguished topography of a protein helps medical chemists design drugs on the basis of the pharmacophoric features of the protein. Structure-based drug discovery, specifically for pathological proteins that cause a higher risk of disease, takes advantage of this fact. Current tools for studying drug-protein interactions include physical, chromatographic, and electrophoretic methods. These techniques can be separated into either non-spectroscopic (equilibrium dialysis, ultrafiltration, ultracentrifugation, etc.) or spectroscopic (Fluorescence spectroscopy, NMR, X-ray diffraction, etc.) methods. These methods, however, can be time-consuming and expensive. On the other hand

Stress management is becoming very important part of cancer patient care. Chronic stressors lead to boost tumorigenesis and promote cancer development, recurrence, and drug resistant leading to poor health outcomes. The Hypothalamic-Pituitary-Adrenal (HPA) axis, which is activated by stress, also regulates Hypothalamic-Pituitary-Thyroid (HPT) axis. Stress related changes in immune function and inflammatory response also leads to reduced immune surveillance resulting in tumorigenesis. This article explores the hormonal axis impacted by stress and how chronic stress can lead to poor outcome of a cancer patient.

Cellular senescence is one of the prevailing issues in cancer therapeutics that promotes cancer relapse, chemoresistance, and recurrence. Patients undergoing persistent chemotherapy often develop drug-induced senescence. Docetaxel, an FDA-approved treatment for prostate cancer, is known to induce cellular senescence which often limits the overall survival of patients. Strategic therapies that counter the cellular and drug-induced senescence are an unmet clinical need. Towards this an effort was made to develop a novel therapeutic strategy that targets and removes senescent cells from the tumors, we developed a nanoformulation of tannic acid−docetaxel self-assemblies (DSAs). The construction of DSAs was confirmed through particle size measurements, spectroscopy, thermal, and biocompatibility studies. This formulation exhibited enhanced in vitro therapeutic activity in various biological functional assays with respect to native docetaxel treatments. Microarray and immunoblot analysis results demonstrated that DSAs exposure selectively deregulated senescence associated TGFβR1/FOXO1/p21 signaling. Decrease in β-galactosidase staining further suggested reversion of drug-induced senescence after DSAs exposure. Additionally, DSAs induced profound cell death by activation of apoptotic signaling through bypassing senescence. Furthermore, in vivo and ex vivo imaging analysis demonstrated the tumor targeting behavior of DSAs in mice bearing PC-3 xenograft tumors. The antisenescence and anticancer activity of DSAs was further shown in vivo by inhibiting TGFβR1 proteins and regressing tumor growth through apoptotic induction in the PC-3 xenograft mouse model. Overall, DSAs exhibited such advanced features due to a natural compound in the formulation as a matrix/binder for docetaxel. Overall, DSAs showed superior tumor targeting and improved cellular internalization, promoting docetaxel efficacy. These findings may have great implications in prostate cancer therapy.

Paclitaxel (PTX) is a gold standard chemotherapeutic agent for breast, ovarian, pancreatic and non-small cell lung carcinoma. However, in clinical use PTX can have adverse side effects or inadequate pharmacodynamic parameters, limiting its use. Nanotechnology is often employed to reduce the therapeutic dosage required for effective therapy, while also minimizing the systemic side effects of chemotherapy drugs. However, there is no nano formulation of paclitaxel with chemosensitization motifs built in. With this objective, we screened eleven pharmaceutical excipients to develop an alternative paclitaxel nanoformulation using a self-assembly method. Based on the screening results, we observed tannic acid possesses unique properties to produce a paclitaxel nanoformulation, i.e., tannic acid-paclitaxel (TAP). This stable TAP nanoformulation, referred to as TAP nanoparticles (TAP NPs), showed a spherical shape of 102.22±14.05 nm and negative zeta potential of −8.85±0.44 mV. The presence of PTX in TAP NPs was confirmed by Fourier Transform Infrared (FTIR) spectra, thermogravimetric analyzer (TGA) scans, and X-ray diffraction (XRD). Encapsulation efficiency of PTX in TAP NPs was determined to be > 96.49±0.43 %. Intracellular drug uptake of plain drug PTX on breast cancer cells (MDA-MB-231) shows more or less constant drug concentration from 2–6 hours, suggesting drug efflux by the P-gp transporters, over TAP NPs, in which PTX uptake was more than 95.52+11.01% in 6 hours, as analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Various biological assays such as proliferation, clonogenic formation, invasion, and migration confirm superior anticancer effects of TAP NPs over plain PTX at all tested concentrations. P-gp expression Rhodamine-123 (RH123), beta-tubulin stabilization, Western blot, and microarray analysis further confirm the improved therapeutic potential of TAP NPs. These results suggest that the TAP nanoformulation provides an important reference for developing a therapeutic nanoformulation affording pronounced, enhanced effects in breast cancer therapy.

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the United States. About 90% of all cancer-related deaths are due to the development of metastatic sites in the body. 40-50% of colorectal cancer patients develop metastasis at some point during their fight with the disease. Understanding the mechanism of metastasis in colorectal cancer is vital. Metastasis is a multistep process; anchorage-independent survival after intravasation of cells from the primary tumor site is a crucial step. In our lab, we have previously demonstrated that MUC13 plays an essential role in the CRC progression by modulating anti-apoptotic pathways and survival proteins expression. Under anchorage-independent growth conditions, we have identified that MUC13, in correspondence with YAP1, is the key upregulated protein and is responsible for increased survival. Isogenic CRC cell lines SW480 (non-metastatic) and SW620 (metastatic) on low adherence growth conditions are applied in these studies. In addition, lentiviral transduced stable overexpression and knockdown cell lines were generated for MUC13 and YAP1 mechanistic studies. The overexpression of MUC13 in non-metastatic SW480 cells (low MUC13 expressing) increased anchorage-independent survival and enhanced tumorigenesis compared to SW480+Vector cells, contrary results were found upon MUC13 knockdown in SW620 cells (high MUC13 expressing). In vitro results were recapitulated in the in vivo mouse model system and human CRC tissues. In Proximity Ligation Assays (PLA), we found an increased nuclear localization of the survival complex YAP1/β-catenin in MUC13 overexpressing cells. In contrast, MUC13 knockdown resulted to the lower aboudance of survival complex in the nucleus. Immunoprecipitation validates the protein-protein interaction between MUC13 and YAP1. YAP1 knockdown in MUC13 overexpressing cells showed a decrease in survival, indicating the necessary functional complex formation between MUC13 and YAP1. MUC13 and YAP1 expression in human CRC tissue were highest at Stage II. However, YAP1 expression increased when MUC13 was observed in the nucleus. This supports the notion that MUC13 is critical in enhancing CRC metastasis through molecular interaction with YAP1. For the first time, this study demonstrates complex formation between MUC13 and YAP1 and defines their role in CRC progression and metastasis. Citation Format: Kyle Doxtater, Manish K. Tripathi, Radhika Sekhri, Sudhir Kotnala, Bilal Hafeez, Sheema Khan, Nadeem Zafar, Murali Yallapu, Meena Jaggi, Subhash Chauhan. MUC13 enhances colorectal cancer metastasis through molecular interaction with YAP1 transcription factor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 992.

Exosomes are nanoscale vesicles generated by cells for intercellular communication. Due to their composition, significant research has been conducted to transform these particles into specific delivery systems for various disease states. In this review, we discuss the common isolation and loading methods of exosomes, some of the major roles of exosomes in the tumor microenvironment, as well as discuss recent applications of exosomes as drug delivery vessels and the resulting clinical implications.

There is increasing evidence suggesting the role of microbiome alterations in relation to pancreatic adenocarcinoma and tumor immune functionality. However, molecular mechanisms of the interplay between microbiome signatures and/or their metabolites in pancreatic tumor immunosurveillance are not well understood. We have identified that a probiotic strain (Lactobacillus casei) derived siderophore (ferrichrome) efficiently reprograms tumor-associated macrophages (TAMs) and increases CD8 + T cell infiltration into tumors that paralleled a marked reduction in tumor burden in a syngeneic mouse model of pancreatic cancer. Interestingly, this altered immune response improved anti-PD-L1 therapy that suggests promise of a novel combination (ferrichrome and immune checkpoint inhibitors) therapy for pancreatic cancer treatment. Mechanistically, ferrichrome induced TAMs polarization via activation of the TLR4 pathway that represses the expression of iron export protein ferroportin (FPN1) in macrophages. This study describes a novel probiotic based molecular mechanism that can effectively induce anti-tumor immunosurveillance and improve immune checkpoint inhibitors therapy response in pancreatic cancer.

Long noncoding RNAs (lncRNAs) are a class of noncoding RNA, involved in regulation of diverse physiological and pathological processes. Ovarian cancer is the leading cause of death among all gynecological malignancies in the world and its underlying mechanism is still unclear. LncRNAs exhibit multiple biological functions in various stages of ovarian cancer development. We will discuss and summarize the new and important lncRNAs and their involvement in disease, which might represent promising therapeutic targets. Therapeutic intervention based on silencing or functional inhibition of target lncRNAs will be beneficial for ovarian cancer patients.

In todays Service-Oriented Architecture (SOA) world, software systems are built by composing web services offered by Service Providers (SPs). There are different SPs offering services for the same set of functional requirements. Service providers are expected to be highly competitive in their offerings to enhance their market. The quality of web services is an important factor that differentiates one service provider from another. Twelve parameters are identified by which quality of service can be measured. The prediction of these twelve QoS parameters help SPs to enhance the quality of their service. Each web service is realized by several programming files. CK and object oriented metrics of the underlying Java files of the web services are important features for predicting QoS parameters of the web service. The aggregated measure, mean, is chosen to be a feature in predicting the QoS parameters in earlier studies. We propose to build prediction models using 16 aggregate measures and show that there is significant difference between these aggregate measures. We find best feature subset using six feature selection techniques and build prediction models using Extreme Learning Machines with different kernels. We show that feature selection techniques might not enhance prediction accuracies and the ensemble algorithm out performs other learning algorithms.

Background: Colorectal carcinoma (CRC) is the second leading cause of cancer related deaths in the United States. The five-year survival rate of patients diagnosed with distant stages declines to 14%, which is of concern when compared to 90% for localized-stage and 71% for regional stage disease. This necessitates the need for early diagnostic biomarkers, to minimize poor drug response/resistance, recurrence, metastasis and mortality. Disproportionate biochemical stressors increase the risk of developing CRC and its progression by influencing molecular drivers within the coding and noncoding parts of the genome. Thus, understanding the biological mechanism of these stress factors on the molecular drivers of this disease can provide pivotal information pertinent to CRC development and progression. Recently, our laboratory has identified a novel long noncoding RNA (lncRNA) namely, Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1), which is highly over-expressed in CRC and is involved in its pathogenesis and is regulated by transcription factor Nuclear Factor of Activated T cell 1 (NFATc1). Methods: Archived human CRC tissues were stained using a recently standardized Z-probe technology. TCGA database of ~600 CRC patients was also analyzed using the bioinformatic approach. CRC cell lines were profiled for MALAT1 expression using RT-PCR. Lentiviral constructs were used to generate stable lncRNA MALAT1 expressing cell lines. CRISPR/Cas9 constructs were used to knockdown lncRNA MALAT1 and NFATc1. Mouse model was used to verify the stress induced expression of MALAT1 and NFATc1. ReCLP (Reversible Cross-Linked Precipitation) and iRAP (invitro RNA Antisense Proteomics) studies are in progress to identify the associated proteins and complexes. Results: RNAScope analysis showed MALAT1 to be highly over-expressed in human CRC tissues. MALAT1 expression increased with stage and negatively correlated with the tumor size. TCGA database analysis confirmed our findings. Multiple NFATc1 binding site were identified by ChIPseq database. Overexpression of transcription factor NFATc1 upregulated lncRNA MALAT1 expression. CRISPR/Cas9 based knockdown of NFATc1 downregulated NFATC1 and lncRNA MALAT1, but vice versa was not true, indicating NFATc1 to be upstream of lncRNA MALAT1. Also, expression of MALAT1, NFATc1 and IL-6 were highly upregulated by biochemical stressor cortisol in mouse colonic tissues. Further studies are in progress for direct association of NFATc1 on MALAT1 promoter and mechanism by which the stress factor regulate NFATc1 expression and hence lncRNA MALAT1 expression. Conclusion: This study helps to understand influence of biochemical stress factors on long noncoding RNA MALAT1 and transcription factor NFATc1 etiology. Early diagnosis of these molecular markers will help in designing novel preventive/therapeutic strategies to reduce CRC progression, metastasis and hence mortality. Citation Format: Kyle Doxtater, Chidi Zacheaus, Radhika Sekhri, Utkarsh K. Mishra, Zachary E. Stiles, Nitish Mishra, Chittibabu Guda, Nadeem Zafar, Mahul Amin, Pradeep Shukla, Murali M. Yallapu, Meena Jaggi, Subhash C. Chauhan, Manish K. Tripathi. Stress regulated role of lncRNA Malat1 in colorectal cancer progression and metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3142.

Formalin-fixed paraffin embedded (FFPE) tissues are a valuable resource for biomarker discovery in order to understand the etiology of different cancers and many other diseases. Proteins are the biomarkers of interest with respect to FFPE tissues as RNA degradation is the major challenge in these tissue samples. Recently, non-protein coding transcripts, long non-coding RNAs (lncRNAs), have gained significant attention due to their important biological actions and potential involvement in cancer. RNA sequencing (RNA-seq) or quantitative reverse transcription-polymerase chain reaction (qRT-PCR) are the only validated methods to evaluate and study lncRNA expression and neither of them provides visual representation as immunohistochemistry (IHC) provides for proteins. We have standardized and are reporting a sensitive Z probe based in situ hybridization method to visually identify and quantify lncRNA in FFPE tissues. This assay is highly sensitive and identifies transcripts visible within different cell types and tumors. We have detected a scarcely expressed tumor suppressor lncRNA NRON (non-coding repressor of nuclear factor of activated T-cells (NFAT)), a moderately expressed oncogenic lncRNA UCA1 (urothelial cancer associated 1), and a highly studied and expressed lncRNA MALAT1 (metastasis associated lung adenocarcinoma transcript 1) in different cancers. High MALAT1 staining was found in colorectal, breast and pancreatic cancer. Additionally, we have observed an increase in MALAT1 expression in different stages of colorectal cancer.

p120-Catenin (p120) functions as a tumor suppressor in intestinal cancer, but the mechanism is unclear. Here, using conditional p120 knockout in Apc-sensitized mouse models of intestinal cancer, we have identified p120 as an "obligatory" haploinsufficient tumor suppressor. Whereas monoallelic loss of p120 was associated with a significant increase in tumor multiplicity, loss of both alleles was never observed in tumors from these mice. Moreover, forced ablation of the second allele did not further enhance tumorigenesis, but instead induced synthetic lethality in combination with Apc loss of heterozygosity. In tumor-derived organoid cultures, elimination of both p120 alleles resulted in caspase-3-dependent apoptosis that was blocked by inhibition of Rho kinase (ROCK). With ROCK inhibition, however, p120-ablated organoids exhibited a branching phenotype and a substantial increase in cell proliferation. Access to data from Sleeping Beauty mutagenesis screens afforded an opportunity to directly assess the tumorigenic impact of p120 haploinsufficiency relative to other candidate drivers. Remarkably, p120 ranked third among the 919 drivers identified. Cofactors α-catenin and epithelial cadherin (E-cadherin) were also among the highest scoring candidates, indicating a mechanism at the level of the intact complex that may play an important role at very early stages of of intestinal tumorigenesis while simultaneously restricting outright loss via synthetic lethality.

Ormeloxifene is a clinically approved selective estrogen receptor modulator, which has also shown excellent anticancer activity, thus it can be an ideal repurposing pharmacophore. Herein, we report therapeutic effects of ormeloxifene on prostate cancer and elucidate a novel molecular mechanism of its anticancer activity. Ormeloxifene treatment inhibited epithelial-to-mesenchymal transition (EMT) process as evident by repression of N-cadherin, Slug, Snail, vimentin, MMPs (MMP2 and MMP3), β-catenin/TCF-4 transcriptional activity, and induced the expression of pGSK3β. In molecular docking analysis, ormeloxifene showed proficient docking with β-catenin and GSK3β. In addition, ormeloxifene induced apoptosis, inhibited growth and metastatic potential of prostate cancer cells and arrested cell cycle in G0–G1 phase via modulation of cell-cycle regulatory proteins (inhibition of Mcl-1, cyclin D1, and CDK4 and induction of p21 and p27). In functional assays, ormeloxifene remarkably reduced tumorigenic, migratory, and invasive potential of prostate cancer cells. In addition, ormeloxifene treatment significantly (P < 0.01) regressed the prostate tumor growth in the xenograft mouse model while administered through intraperitoneal route (250 μg/mouse, three times a week). These molecular effects of ormeloxifene were also observed in excised tumor tissues as shown by immunohistochemistry analysis. Our results, for the first time, demonstrate repurposing potential of ormeloxifene as an anticancer drug for the treatment of advanced stage metastatic prostate cancer through a novel molecular mechanism involving β-catenin and EMT pathway. Mol Cancer Ther; 16(10); 2267–80. ©2017 AACR.

Background Lung cancer is the leading cause of cancer deaths across globe. Statistically, 70-80% of lung cancer cases fall under non-small cells lung cancer (NSCLC) category. Recently, usage of natural compounds (e.g. EGCG, curcumin, resveratrol, apigenin, etc.,) for cancer therapy has gained lots of attention. However, poor dissolution profiles of these natural compounds lead to unfavorable pharmacological doses and administration. For this purpose, we have candidate, a water-soluble natural polyphenol, Tannic acid (TA, C76H52O46), which, a natural polyphenol that is richly found in plants such as green tea. This molecule exhibits anti-oxidant and anti-proliferative activities, which is being used as an alternative therapeutic option in cancer therapy. However, role of TA in NSCLC was not examined. Therefore, the aim of present study is to assess the molecular effects of TA in NSCLC cells. Methods For this study, two NSCLC cell lines (A549 and H1299) and one normal lung cell line (BEAS-2B) were used. We investigated the anti-proliferative effects of TA on NSCLC cells using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) tetrazolium (MTT), and colony formation assays. The anti-invasiveness and anti-migratory potential of TA was evaluated through Matrigel and Boyden chamber studies, respectively. Cell cycle and apoptotic effects of TA was evaluated through flow cytometry and western blot studies. The quantification of secreted VEGF was assessed by ELISA. Also, for affirmation results of gene expression profiles we performed q-PCR studies. Results Cell proliferation study demonstrated a dose dependent anti-cancer effects of TA on NSCLC cells. However, TA had no significant toxicity effects on human bronchial epithelial cells. Similarly, colony formation results exhibited dose dependence reduction in response to escalated dose of TA exposure. Both the invasion and migration results confirm its anti-metastatic role. Extracellular VEGF quantification showed lower VEGF secretion with TA treatments compared to the control cells. The cell cycle analysis evidently showed TA efficiently induced G1 phase cell arrest in NSCLC cells which was further confirmed through western blotting studies of relevant proteins (Cyclin D1, P53, p21, p18, BAX, BCL-2). We also achieved to show the decreased expression of VEGFR2, p-AKT, and cleaved caspase 7 indicating TA induces apoptosis. Conclusion Overall, these results demonstrate illustrate anti-cancer activity of TA by targeting the VEGF related pathways in NSCLC. This approach can lead to an alternative therapeutic strategy for lung cancer, with ideal PK/PD profiles. Citation Format: Elham Hatami, Prashanth K.B. Nagesh, Pallabita Chowdhury, Advit Bhaskar Shetty, Manish K. Tripathi, Subhash Chauhan, Meena Jaggi, Murali Yallapu. Tannic acid: A natural anticancer agent for non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1871.

Purpose: Prostate cancer (PrCa) is one of the leading causes of cancer-related deaths among men in the United States. Chemotherapy with docetaxel holds great promise and important therapeutic option for PrCa treatment. However, chemotherapy is often associated with incomplete cell death and leads to persistent senescent phenotype. Such phenotypic cancer cells survive and promotes tumor development, recurrence, and drug resistance. Altogether, targeting these cells can enhance chemotherapy outcomes. In the current study, we established a tannic acid-docetaxel scaffold nanoparticles (TDS NP) platform that allows for facile targeting and inhibition of drug induced senescence in prostate cancer. Methods: TDS NP were prepared by solvent evaporation and extrusion process using a series of tannic acid and docetaxel weight ratios. The scaffold self-assembly formation was determined using a steady-state fluorescence quenching, FT-IR, XRD, DSC, and TGA. The surface and morphological properties of TDS NPs were determined by dynamic light scattering and transmission electron microscopy. The biocompatibility assessment was characterized by hemolysis assay. C4-2 and PC-3 cell lines were used as PrCa model systems for in vitro and in vivo studies. The cellular internalization (in vitro) and accumulation (in vivo) was examined using dye-tagged TDS NPs. The superior in vitro anti-cancer and metastatic potential of TDS NPs were evaluated using proliferation, colony formation, migration, invasion, and immunoblotting assays. The anti-senescence ability of TDS NPs was examined through β-Galactosidase Staining Kit. A PC-3 xenograft mouse model was used to examine its superior therapeutic activity over free docetaxel treatment. Results: A series of physico-chemical analyses confirmed the integrity of TDS NP. An optimized formulation exhibited a spherical shape particle formulation with 124.13 ± 2.16 nm with a negative zeta potential of -14.0 mV. TDS NPs formulation exhibited superior internalization capacity in PrCa cells in a dose- and time-dependent manner. In vitro functional studies confirm superior therapeutic activity of TDS NPs over free docetaxel treatment. Enhanced pro-apoptotic while downregulating anti-apoptotic effects with treatment of TDS NPs in comparison to native drug. A profound inhibition of β-galactosidase activity was noticed with TDS NPs treatment. In vivobiodistribution studies confirm efficient accumulation of TDS NPs in mice. In addition, TDS NPs showed robust antitumor activity against PC-3 xenografts. Such improved activity was correlated with increased inhibition of senescence and induced apoptosis. Conclusion: In summary, we developed a novel tannic acid-docetaxel scaffold nanoparticle formulation that is capable of inhibiting senescence, minimizing drug resistance, and inducing apoptosis in prostate cancer. Citation Format: Prashanth Kumar Bhusetty Nagesh, Pallabita Chowdhury, Elham Hatami, Sonam Kumari, Vivek Kumar Kashyap, Bilal Hafeez, Sheema Khan, Manish Kumar Tripathi, Meena Jaggi, Subhash C. Chauhan, Murali M. Yallapu. Tannic acid-docetaxel scaffold nanoparticles for improved prostate cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3634.

Objective: Colorectal cancer (CRC) is a leading cause of cancer mortality affecting over a million people every year. Biological markers for early detection and distant metastatic disease in patients with CRC are not well defined. We have identified transmembrane mucin MUC13, which is expressed in normal colon mucosa to be highly expressed in colorectal cancer, but the underlying pathways and the signaling mechanisms involved in CRC pathogenesis are not known. Our studies suggest MUC13 correlation with demographic and clinicopathologic characteristics. Materials and Methods: Retrospective institutional tumor registry was reviewed to identify patients with resected colon adenocarcinoma. Archived FFPE tissue blocks were reviewed by an experienced pathologist. Selected representative tissue blocks were serially sectioned at 4 µm. IHC staining was performed using an in-house MAb for MUC13. Slides were digitally scanned and analyzed qualitatively as well as quantitatively using a modified H-score based on the intensity of expression and percentage of stained cells. MUC13 was correlated with disease stage, aggressiveness and inflammatory markers, indicating poor prognosis in CRC patients. MUC13 splice variants were probed using novel designed probes. MUC13 SNPs were identified using unbiased approach by analyzing dpGAP database. Results: 196 tissues, of which 56.1% were female, 52% were white, and the median age at resection was 70. 38 (19.4%) were stage I, 64 (32.7%) stage II, 84 (42.9%) stage III, and 10 (5.1%) stage IV. 100% of colon adenocarcinoma tissues stained positively for MUC13, including definitive tumor epithelial staining, little-to-no background stromal staining, and mild staining of adjacent normal colon mucosa. Typical colon adenocarcinoma cells exhibited strong apical membranous staining with varying degrees of cytoplasmic staining. Advanced stage tumors were noted to more frequently exhibit basolateral and/or circumferential membranous staining compared to early stage tumors which more frequently displayed apical membrane staining alone. Five MUC13 transcript variants were identified by database analysis. Two of the protein coding MUC13 variants (long and short) showed differential expression in aggressive cell lines and patient tissues. Cyclic turnover between the short and long isoforms were observed during Anoikis resistance, cortisol, alcohol and BAP treatments. SNPs have been associated with increased risk of cancer incidence or fatality. Relevant MUC13 SNPs have been identified through systematic analysis of patient databases (dpGAP database). Verification and correlation of identified SNPs with patient disease stage and prognosis will also be presented. Conclusion: This is the first study to correlate MUC13 expression with disease outcome. Alternative transcripts and single nucleotide polymorphisms gave genetic insight of the role of MUC13 in CRC pathogenesis. Citation Format: Manish K. Tripathi, Chidi Zacheaus, Kyle Doxtater, Zachary Stiles, Fatemeh Keramatnia, Nadeem Zafar, Mahul Amin, Meena Jaggi, Subhash Chauhan. MUC13 is a novel molecular signature, for early detection and metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5178.

International audience; The general transcription factor TFIID is a large multisubunit complex required for the transcription of most protein-encoding genes by RNA polymerase II. Taking advantage of a TFIID preparation partially depleted in the initiator-binding Taf2p subunit, we determined the conformational and biochemical variations of the complex by electron tomography and cryo-electron microscopy of single molecules. Image analysis revealed the extent of conformational flexibility of the complex and the selection of the most homogeneous TFIID subpopulation allowed us to determine an improved structural model at 23 Angstroms resolution. This study also identified two subpopulations of Taf2p-containing and Taf2p-depleted TFIID molecules. By comparing these two TFIID species we could infer the position of Taf2p, which was confirmed by immunolabeling using a subunit-specific antibody. Mapping the position of this crucial subunit in the vicinity of Taf1p and of TBP sheds new light on its role in promoter recognition.

Replication of kDNA in the mitochondrion of the kinetoplastid protozoan is an essential process. One of the proteins that may be required for the kDNA replication is the ribonuclease H (RNase H; EC 3.1.26.4). We have identified four distinct ribonuclease H genes in Leishmania, one type I (LRNase HI) and three type II (LRNase HIIA, LRNase HIIB and LRNase HIIC). We detail here molecular characterization of LRNase HIIC. The coding sequence of LRNase HIIC is 1425 bp in length encoding a 474-amino acid protein with a calculated molecular mass of approximately 53 kDa. While LRNase HIIC shares several conserved domains with mitochondrial RNase H from other organisms, it has three extra patches of amino acid sequences unique to this enzyme. Functional identity of this protein as an RNase H was verified by genetic complementation in RNase H-deficient Escherichia coli. The precursor protein may be enzymatically inactive as it failed to complement the E. coli mutant. The mitochondrial localization signal in LRNase HIIC is within the first 40 amino acid residues at the N-terminus. In vitro import of the protein by the mitochondrial vesicles showed that the precursor protein is processed to a 49-kDa protein. Antisense ablation of LRNase HIIC gene expression is lethal to the parasite cells both in vitro and in vivo. This study not only reveals the significance of the LRNase HIIC in the kinetoplast biology but also identifies a potential molecular target for antileishmanial chemotherapy.

The parasitic protozoan Leishmania specifically manipulates the expression of host macrophage genes during initial interactions, as revealed by mRNA differential display reverse transcription-PCR and cDNA microarray analyses. The genes that are down-regulated in mouse (J774G8) or human (U937) macrophages upon exposure to Leishmania include small RNA transcripts from the short interspersed element sequences. Among the short interspersed element RNAs that are down-regulated is 7SL RNA, which is the RNA component of the signal recognition particle. Because the microbicidal functions of macrophages profoundly count on vesicular protein transport processes, down-regulation of 7SL RNA may be significant in the establishment of infection by Leishmania in macrophage phagolysosomes. To evaluate whether down-regulation of 7SL RNA results in inhibition of signal recognition particle-mediated vesicular protein transport processes, we have tested and found that the targeting of proteins to the endoplasmic reticulum and plasma membrane and the secretion of proteins by macrophages are compromised in Leishmania-infected J774G8 and U937 cells. Knocking down 7SL RNA using small interfering RNA mimicked the effect of exposure of macrophages to Leishmania. The overexpression of 7SL RNA in J774G8 or U937 cells made these cells resistant to Leishmania infection, suggesting the possible biological significance of down-regulation of 7SL RNA synthesis in the establishment of infection by Leishmania. We conclude that Leishmania down-regulates 7SL RNA in macrophages to manipulate the targeting of many proteins that use the vesicular transport pathway and thus favors its successful establishment of infection in macrophages.

Invasiveness of tumor cells is often determined by the profile of their expressed genes. To determine the gene expression differences between an invasive and a non-invasive human breast tumor cells, we selected BT-549 (invasive) and MDA-MB-468 (non-invasive) cells, and compared their transcriptomes by cDNA microarray analysis. Among the significant differences in gene expressions, notable are the up-regulation of cytokeratins 8 and 19, and down-regulation of metallothioneins 1G and IL in MDA-MB-468 cells. Since MDA-MB-468 cells do not express SLUG, a member of a small family of E2-box-binding zinc finger silencer proteins, we studied whether the cytokeratin gene overexpressions in these cells are due to the absence of SLUG. Inducible expression of SLUG in MDA-MB-468 cells inhibited the expressions of the cytokeratin 8 and 19 but not others as was revealed by microarray analysis. Similarly, siRNA knock down of SLUG in BT-549 cells increased the expressions of those cytokeratin mRNAs. SLUG levels in the cell regulated the function of cytokeratins 8 and 19 gene promoters. We conclude that the expressions of cytokeratins and metallothioneins may be associated with the differential invasive behaviors of these breast tumor cells and SLUG may have regulatory roles in this process.

To understand the effects of the transient ablation of BRCA2 gene expression in dividing human breast cells, we transiently knocked down BRCA2 mRNA in HMEC and other cells. Microarray analysis of mRNAs revealed the down-regulation of the mRNAs of ubiquitin cross-reacting protein (UCRP) and the E2 enzyme that help conjugating UCRP to its target proteins, namely UBE2L6 (UbcH8), in BRCA2 ablated cells. UCRP is an interferon regulated protein, involved in cell growth and cell cycle events by participating in the degradation/modulation of cell cycle regulatory proteins. Quantitative-PCR and Northern analysis confirmed down-regulation of UCRP and UBE2L6 with BRCA2 knockdown, respectively. Since UCRP and UCRPylation have critical roles in the innate immunity against viral infection and during pregnancy, our observation may indicate new roles of the BRCA2 protein.

An expression library generated from genomic DNA of Streptococcus sp. N1 resulted in C1N1 Escherichia coli recombinants forming haloes on lipase indicator tributyrin/Tween-80 agar plates. The excised insert in the recombinant C1N1 was of 3.489 kb size. Analysis of the nucleotide sequence of the insert showed two significant operons of 1032 bp (ORF1) and 729 bp (ORF3). ORF1 on the (+)-strand is the structural lipase gene codes for a protein of 343 amino acid residues with a theoretical mass of 38.6 kDa, which is close to the value of the lipase expressed in E. coli (39.36 kDa). The primary structure of the lipase deduced from the nucleotide sequence shows a consensus sequence containing the active serine [VAGHSIGG], a conserved H–G dipeptide in the N-terminal part of the enzyme and a potential site for N-linked glycosylation at amino acid residues [129–131]. The sequence, AAGGA at −6 upstream of the start codon ATG seems to be putative ribosomal binding site (rbs) and is similar to the rbs of lip3 of Moraxella sp. TA 144. The putative −10[TTTAT] and −35[AATG] promoter sequences showed similarity to lip3 of Moraxella sp. TA 144. The lipase shared no significant homology with the members of different lipases families except with group V comprising of Psychrobacter immobilis B-10 [28% identical and 56% positive similarity] and Moraxella sp. TA 144, lip3 [28% identical and 54% similarities]. The 729 bp operon present on (−) strand of 3.489 kb C1N1 insert was essential for the secretion of the enzyme. The putative protein on BLAST/FASTA search showed 57% identities and 72% positives with ATP-binding component of ABC-transporter. The recombinant lipase was resistant to organic solvents, non-ionic surfactants, metal ions and showed alkaline pH optima (pH 8.4).