Gaurav Kaushik

Honokiol inhibits melanoma stem cells by targeting notch signaling

Melanoma is an aggressive disease with limited therapeutic options. Here, we determined the effects of honokiol (HNK), a biphenolic natural compound on melanoma cells and stemness. HNK significantly inhibited melanoma cell proliferation, viability, clonogenicity and induced autophagy. In addition, HNK significantly inhibited melanosphere formation in a dose dependent manner. Western blot analyses also demonstrated reduction in stem cell markers CD271, CD166, Jarid1b, and ABCB5. We next examined the effect of HNK on Notch signaling, a pathway involved in stem cell self‐renewal. Four different Notch receptors exist in cells, which when cleaved by a series of enzymatic reactions catalyzed by Tumor Necrosis Factor‐α‐Converting Enzyme (TACE) and γ‐secretase protein complex, results in the release of the Notch intracellular domain (NICD), which then translocates to the nucleus and induces target gene expression. Western blot analyses demonstrated that in HNK treated cells there is a significant reduction in the expression of cleaved Notch‐2. In addition, there was a reduction in the expression of downstream target proteins, Hes‐1 and cyclin D1. Moreover, HNK treatment suppressed the expression of TACE and γ‐secretase complex proteins in melanoma cells. To confirm that suppression of Notch‐2 activation is critical for HNK activity, we overexpressed NICD1, NICD2, and performed HNK treatment. NICD2, but not NICD1, partially restored the expression of Hes‐1 and cyclin D1, and increased melanosphere formation. Taken together, these data suggest that HNK is a potent inhibitor of melanoma cells, in part, through the targeting of melanoma stem cells by suppressing Notch‐2 signaling.

Honokiol induces cytotoxic and cytostatic effects in malignant melanoma cancer cells

BACKGROUND Melanomas are aggressive neoplasms with limited therapeutic options. Therefore, developing new therapies with low toxicity is of utmost importance. Honokiol is a natural compound that recently has shown promise as an effective anticancer agent. METHODS The effect of honokiol on melanoma cancer cells was assessed in vitro. Proliferation and physiologic changes were determined using hexosaminidase assay and transmission electron microscopy. Protein expression was assessed by immunoblotting. RESULTS Honokiol treatment inhibited cell proliferation and induced death. Electron microscopy showed autophagosome formation. Reduced levels of cyclin D1 accompanied cell-cycle arrest. Honokiol also decreased phosphorylation of AKT (known as protein kinase B) and mammalian target of rapamycin, and inhibited γ-secretase activity by down-regulating the expression of γ-secretase complex proteins, especially anterior pharynx-defective 1. CONCLUSIONS Honokiol is highly effective in inhibiting melanoma cancer cells by attenuating AKT/mammalian target of rapamycin and Notch signaling. These studies warrant further clinical evaluation for honokiol alone or with present chemotherapeutic regimens for the treatment of melanomas.

Natural compounds targeting major cell signaling pathways: a novel paradigm for osteosarcoma therapy

Osteosarcoma is the most common primary bone cancer affecting children and adolescents worldwide. Despite an incidence of three cases per million annually, it accounts for an inordinate amount of morbidity and mortality. While the use of chemotherapy (cisplatin, doxorubicin, and methotrexate) in the last century initially resulted in marginal improvement in survival over surgery alone, survival has not improved further in the past four decades. Patients with metastatic osteosarcoma have an especially poor prognosis, with only 30% overall survival. Hence, there is a substantial need for new therapies. The inability to control the metastatic progression of this localized cancer stems from a lack of complete knowledge of the biology of osteosarcoma. Consequently, there has been an aggressive undertaking of scientific investigation of various signaling pathways that could be instrumental in understanding the pathogenesis of osteosarcoma. Here, we review these cancer signaling pathways, including Notch, Wnt, Hedgehog, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT, and JAK/STAT, and their specific role in osteosarcoma. In addition, we highlight numerous natural compounds that have been documented to target these pathways effectively, including curcumin, diallyl trisulfide, resveratrol, apigenin, cyclopamine, and sulforaphane. We elucidate through references that these natural compounds can induce cancer signaling pathway manipulation and possibly facilitate new treatment modalities for osteosarcoma.

Targeting Cancer Stem Cells for Chemoprevention of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma is one of the deadliest cancers worldwide and the fourth leading cause of cancer-related deaths in United States. Regardless of the advances in molecular pathogenesis and consequential efforts to suppress the disease, this cancer remains a major health problem in United States. By 2030, the projection is that pancreatic cancer will be climb up to be the second leading cause of cancer-related deaths in the United States. Pancreatic cancer is a rapidly invasive and highly metastatic cancer, and does not respond to standard therapies. Emerging evidence supports that the presence of a unique population of cells called cancer stem cells (CSCs) as potential cancer inducing cells and efforts are underway to develop therapeutic strategies targeting these cells. CSCs are rare quiescent cells, and with the capacity to self-renew through asymmetric/symmetric cell division, as well as differentiate into various lineages of cells in the cancer. Studies have been shown that CSCs are highly resistant to standard therapy and also responsible for drug resistance, cancer recurrence and metastasis. To overcome this problem, we need novel preventive agents that target these CSCs. Natural compounds or phytochemicals have ability to target these CSCs and their signaling pathways. Therefore, in the present review article, we summarize our current understanding of pancreatic CSCs and their signaling pathways, and the phytochemicals that target these cells including curcumin, resveratrol, tea polyphenol EGCG (epigallocatechin- 3-gallate), crocetinic acid, sulforaphane, genistein, indole-3-carbinol, vitamin E δ- tocotrienol, Plumbagin, quercetin, triptolide, Licofelene and Quinomycin. These natural compounds or phytochemicals, which inhibit cancer stem cells may prove to be promising agents for the prevention and treatment of pancreatic cancers.

Rabix: An Open-Source Workflow Executor Supporting Recomputability and Interoperability of Workflow Descriptions.

As biomedical data has become increasingly easy to generate in large quantities, the methods used to analyze it have proliferated rapidly. Reproducible and reusable methods are required to learn from large volumes of data reliably. To address this issue, numerous groups have developed workflow specifications or execution engines, which provide a framework with which to perform a sequence of analyses. One such specification is the Common Workflow Language, an emerging standard which provides a robust and flexible framework for describing data analysis tools and workflows. In addition, reproducibility can be furthered by executors or workflow engines which interpret the specification and enable additional features, such as error logging, file organization, optim1izations to computation and job scheduling, and allow for easy computing on large volumes of data. To this end, we have developed the Rabix Executor, an open-source workflow engine for the purposes of improving reproducibility through reusability and interoperability of workflow descriptions.

The Cancer Genomics Cloud: Collaborative, Reproducible, and Democratized—A New Paradigm in Large-Scale Computational Research

The Seven Bridges Cancer Genomics Cloud (CGC; www.cancergenomicscloud.org) enables researchers to rapidly access and collaborate on massive public cancer genomic datasets, including The Cancer Genome Atlas. It provides secure on-demand access to data, analysis tools, and computing resources. Researchers from diverse backgrounds can easily visualize, query, and explore cancer genomic datasets visually or programmatically. Data of interest can be immediately analyzed in the cloud using more than 200 preinstalled, curated bioinformatics tools and workflows. Researchers can also extend the functionality of the platform by adding their own data and tools via an intuitive software development kit. By colocalizing these resources in the cloud, the CGC enables scalable, reproducible analyses. Researchers worldwide can use the CGC to investigate key questions in cancer genomics. Cancer Res; 77(21); e3-6. ©2017 AACR.

Abstract 1893: Honokiol prevents colonic tumorigenesis and affects stem cell viability by affecting oncogenic YAP1 function

Background: Despite advances in early detection, colon cancer remains the second leading cause of death in the United States. We are focused on developing dietary prevention strategies. HNK (HNK) is a biphenolic compound that is used in the traditional Chinese Medicine for treating various ailments. The current study is designed to determine whether HNK affected colon cancer stem cells and to identify a mechanism. Method: Colon cancer (CRC) cell lines HCT116 and SW480 and normal colon epithelial cells were used in the study. Cell growth was measured by hexoseaminidase and clonogenicity assays. Apoptosis was determined by measuring caspase 3/7 activities. Colosphere formation assay and FACS sorting were used for stem cells. For in vivo effects, we used the AOM/DSS-induced colonic tumorigenesis model. Immunohistochemistry was determined for stem cell markers and Hippo signaling proteins. Results: HNK induced a significant dose-dependent inhibition of proliferation and colony formation of the two CRC lines, but induced apoptosis. HNK did not affect the normal cells. To demonstrate HNK effects on stem cells, we performed colosphere assays. HNK significantly reduced the number and size of colospheres, suggesting effects on stem cells. In addition, colon stem cell marker proteins DCLK1, LGR5, and CD44 were also decreased. Further proof was obtained by flow cytometry analyses, where HNK reduced the number of DCLK1+ cells. We next determined whether stem cell signaling is affected. For this, we looked at the Hippo signaling pathway, which is active in intestinal stem cells. The key effector protein of this pathway, YAP1 is also oncogenic in many cancer types. In the canonical Hippo signaling pathway, YAP1 function is inhibited. When YAP1 is phosphorylated at Ser127 by the action of upstream Mst1/2 and Lats1/2 kinases, it is sequestered in the cytoplasm where it is degraded, thereby inhibiting downstream gene expression. HNK significantly reduced YAP1 levels. Furthermore, HNK inhibited the expression of YAP interacting proteins TEAD1, TEAD2, and TEAD4. On the other hand, ectopic expression of the TEAD1 partially rescued the cells from HNK-mediated growth suppression. To determine the in vivo effect of HNK on AOM/DSS induced colonic tumorigenesis, HNK were oral gavaged at a dose of 5mg/kg bw for 24 weeks. HNK treatment significantly reduced the colonic tumor numbers and size. Western blot and immunohistochemistry analyses demonstrated significant inhibition in the expression of stem marker proteins, oncogenic YAP1 phosphorylation and TEAD1 in the HNK-treated AOM/DSS colonic tumor tissues. Conclusion: Together, these data suggest that HNK prevents colonic tumorigenesis that targets stem cells by inhibiting oncogenic YAP1 in Hippo signaling pathway. Citation Format: Dharmalingam Subramaniam, Sivapriya Ponnurangam, Deep Kwatra, Gaurav Kaushik, Prabhu Ramamoorthy, Satish Ramalingam, Ossama Tawfik, Scott J. Weir, Subhash Padhye, Dan A. Dixon, Shahid Umar, Roy A. Jensen, Shrikant Anant. Honokiol prevents colonic tumorigenesis and affects stem cell viability by affecting oncogenic YAP1 function. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1893. doi:10.1158/1538-7445.AM2015-1893

Using the Seven Bridges Cancer Genomics Cloud to Access and Analyze Petabytes of Cancer Data

Next‐generation sequencing has produced petabytes of data, but accessing and analyzing these data remain challenging. Traditionally, researchers investigating public datasets like The Cancer Genome Atlas (TCGA) would download the data to a high‐performance cluster, which could take several weeks even with a highly optimized network connection. The National Cancer Institute (NCI) initiated the Cancer Genomics Cloud Pilots program to provide researchers with the resources to process data with cloud computational resources. We present protocols using one of these Cloud Pilots, the Seven Bridges Cancer Genomics Cloud (CGC), to find and query public datasets, bring your own data to the CGC, analyze data using standard or custom workflows, and benchmark tools for accuracy with interactive analysis features. These protocols demonstrate that the CGC is a data‐analysis ecosystem that fully empowers researchers with a variety of areas of expertise and interests to collaborate in the analysis of petabytes of data.

Abstract 2595: Enabling petabyte-scale cancer genomics with the NCI Cancer Cloud Pilots

The advent of next generation sequencing has accelerated the generation of genomic data and created a need for methodologies to organize, share, and analyze large volumes of data. To date, petabytes of multi-dimensional information from thousands of patients have been collected. Access and analysis of this information becomes increasingly challenging as the amount of data grows. This difficulty is exemplified when we consider data generated by the efforts of The Cancer Genomics Atlas (TCGA) network, which encompasses more than 2.5 petabytes. Historically, downloading the complete TCGA repository can require several weeks with a highly optimized network connection and access to large institutional compute clusters to perform integrated analysis, which is out of reach for many researchers. The Cancer Cloud Pilot project seeks to directly address these challenges by co-localizing data with the computational resources to analyze it where researchers can access it securely and easily. The project was born out of the recognition that conducting biological research is increasingly computationally-intensive and new approaches are required to support effective data discovery, storage, computation, and collaboration. Funded by the National Cancer Institute, the Cancer Genomics Cloud (CGC) enables researchers to leverage the power of cloud computing to gain actionable insights about cancer biology and human genetics from massive public datasets including TCGA and the Cancer Cell Line Encyclopedia on the CGC. Our approach to create a cancer cloud platform includes collaborative tools, security permissions, data harmonization, and making the data easier to query through the use of metadata curation, resource description frameworks, and visual tools. Additionally, we implemented the Common Workflow Language, an emerging standard for describing computational workflows, to support computational reproducibility. To date, more than 1200 researchers have accessed and analyzed TCGA and analyzed more than 50000 samples on the CGC since its launch in February 2016. In addition to the motivation, inception, and development of the CGC, we will present a case study on the application of unsupervised learning methods to identify individual cell types within tumors using RNA Sequencing data from TCGA cohorts. We will demonstrate how these computationally-intensive methods are benefited by the cloud and how researchers can apply open pipelines to interrogate cancer subtypes and mixed cell populations from TCGA data on their own data. Citation Format: Gaurav Kaushik, Yilong Li, Erik Lehnert, Zeynep Onder, Devin Locke, Brandi N. Davis-Dusenbery, Deniz Kural. Enabling petabyte-scale cancer genomics with the NCI Cancer Cloud Pilots [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2595. doi:10.1158/1538-7445.AM2017-2595

Abstract 3227: Novel Marmelin analog DBQ targets Notch signaling pathway in colon cancer stem cells

Background: Colon cancer is the second leading cause of death in the United States. Previously, we have reported that the identification of a novel compound Marmelin from Aegle marmelos and potent anti-colon cancer activity. We have developed novel Marmelin analogue THB from this structure showed potent anti-cancer activity and its inhibitory constant value was 10 µM. From this, we developed a second series of analogs, of which DBQ is even more potent than THB. The current study is designed to determine whether DBQ affects colon cancer stem cells and identify a mechanism. Method: Colon cancer cell lines HCT116 and SW480 and normal colon epithelial cells were used in the study. Cell growth was measured by hexoseaminidase and clonogenicity assays. Apoptosis was determined by measuring caspase 3/7 activities. Colosphere formation assay and FACS sorting were used for stem cells. For in vivo effects, we performed studies in HCT116 tumor xenografts. Immunohistochemistry was determined for stem cell markers and Notch signaling proteins. Results: DBQ treatment induced significant dose-dependent inhibition of proliferation and colony formation of HCT116 and SW480 cells, but not that of the normal FHC colon epithelial cells. DBQ also significantly reduced the number and size of colospheres, suggesting effects on stem cells. In addition, DBQ reduced the levels of colon stem cell marker proteins DCLK1, LGR5, and CD44. We obtained further confirmation by flow cytometry, where DBQ treatment reduced the number of DCLK1+ cells. We next determined whether DBQ affects the Notch signaling, a pathway that is important in maintaining CSC population. Notch receptor and its ligands are up-regulated in human colon cancer tissues. DBQ treatment significantly downregulated the expression of all four Notch isoforms, its ligands Jagged 1, 2 and DLL1, 3, 4 and downstream target protein Hes1. Notch activation requires cleavage by the γ-secretase complex. DBQ treatment inhibits the expression of γ-secretase complex proteins. To confirm that DBQ effect is thorough downregulating Notch activation, we ectopically expressed the Notch Intracellular domain. DBQ effect was significantly mitigated in this condition. To determine the effect of DBQ on tumor growth in vivo, we administered DBQ intraperitoneally (5mg/kg bw) every day for 21 days in mice carrying HCT116 tumor xenografts. DBQ treatment significantly suppressed tumor xenograft growth, with notably lower tumor volume and weight. Western blot and immunohistochemistry analyses demonstrated significant inhibition of CSC marker proteins DCLK1, LGR5 and CD44 and also the Notch signaling proteins in the DBQ-treated xenograft tissues. Conclusion: Together, these data suggest that DBQ treatment suppresses colon cancer growth that targets stem cells in part by inhibiting Notch signaling pathway. Citation Format: Dharmalingam Subramaniam, Sivapriya Ponnurangam, Prasad R. Dandawate, Gaurav Kaushik, Ossama W. Tawfik, Roy A. Jensen, Santimukul Santra, Subhash B. Padhye, Scott J. Weir, Shrikant Anant. Novel Marmelin analog DBQ targets Notch signaling pathway in colon cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3227. doi:10.1158/1538-7445.AM2017-3227