Effects of modified bioactive pectins on colon cancer cells in vitro

Pectin is a complex structural polysaccharide present in the cell walls of terrestrial plants, fruit and vegetables. Modified pectin (MP), pectin treated with pH, heat or enzymes, has been shown to have anti-cancer activity in several cancer cell lines. The galactan chains of MP are postulated to be essential for bioactivity due to their ability to bind and inhibit the pro-metastatic protein galectin-3 (Gal3) on cancer cells. However, the structural requirements for bioactive MP, as well as interactions with Gal3 in vitro, have rarely been addressed. In this study several pectins from citrus, sugar beet and potato were screened for their biological effects on colon cancer cells, their structures characterised in detail to assess the structure-function relationship and the molecular mechanisms of action investigated. Alkali-treated sugar beet pectin (SSBA) reduced viability of HT29 cells via induction of apoptosis. The enzymatic removal of galactan side chains abolished activity indicating their importance for anti-proliferative action. Additionally, potato rhamnogalacturonan I (P-RGI) reduced viability of DLD1 cells and the homogalacturonan backbone, not the galactan side chains, was shown to be essential for bioactivity. siRNA-mediated knockdown of Gal3 expression in cells showed that bioactivities of SSBA and P-RGI are independent of Gal3, prompting an investigation into alternative mechanisms of action. Expression of the adhesion molecule ICAM1 was shown to be significantly reduced by P-RGI, suggesting a novel potential mode of action. Results presented in this thesis suggest that MPs of varying structures can exert anti-proliferative activity in colon cancer cells via Gal3-independent mechanisms and in a cell-specific manner. This study is also the first to report the anti-cancer activity of sugar beet pectin. The structural complexity of pectin makes it a potential multi-functional therapeutic agent, and results highlight the need for extensive structural characterisation of bioactive pectins as well as further exploration of Gal3-independent mechanisms of action

Molecular characterisation of the poorly differentiated and undifferentiated thyroid carcinomas using genome-wide approaches

Tese de doutoramento, Bioquímica (Genética Molecular), Universidade de Lisboa, Faculdade de Ciências, 2013Poorly differentiated (PDTC) and anaplastic thyroid carcinomas (ATC) are highly malignant tumours composed by dedifferentiated cells, for which current therapeutic options have been ineffective. In the present project, the molecular signatures and genetic alterations associated with these tumours were elucidated, by using genome-wide expression analysis as first assessment. The role of the microRNA stability in thyroid tumourigenesis was also evaluated. The comparison of the expression profiles between PDTC, well-differentiated thyroid carcinomas and normal thyroid tissues, and between ATC and normal thyroid tissues, revealed that PDTC and ATC have common deregulated signatures, indicative of cell adhesion impairment and increased cell cycle, proliferation and chromosomal instability. Additionally, ATC were specifically characterized by loss of epithelial and thyroid-related functions and activation of components from TGF-β pathway. The gene expression data suggested that follicular variants of papillary carcinomas were possible precursors of PDTC, whereas, ATC were more similar to classical papillary carcinomas. Validation by quantitative RT-PCR in independent sample sets, allowed to confirm the significant over-expression of UHRF1 (associated to proliferation) and downregulation of ITIH5 (associated to cell adhesion and invasion) in PDTC relatively to normal tissues. Moreover, CDKN3 gene, which encodes a cyclin-dependent kinase inhibitor, was shown to be significantly upregulated in PDTC and ATC relatively to normal thyroid, and in accordance with its suppressive function, CDKN3 was aberrantly spliced in ATC samples. The over-expression of the epithelial-tomesenchymal transition factor SNAI2, which is induced by TGF-β, was significant in ATC. Attending to the deregulated pathways identified, a mutational screening was undertaken in 26 ATC and 22 PDTC, which included the hot-spot regions of RAS, BRAF, TP53, CTNNB1 (β-catenin) and PIK3CA genes, and, for the first time, a comprehensive mutational analysis of cell cycle [CDKN1A (p21CIP1); CDKN1B (p27KIP1); CDKN2A (p14ARF, p16INK4A); CDKN2B (p15INK4B); CDKN2C (p18INK4C)], and cell adhesion regulators (AXIN1). Most mutations were identified in TP53 (42% of ATC; 27% of PDTC) and in RAS (31% of ATC; 18% of PDTC). The alterations in these genes were mutually exclusive (P=0.0354), and were associated with a lower patient survival (P=0.0383). CDKN2A and CDKN2B were found to be mutated, for the first time, in up to 25% of PDTC. Other known recurrent mutations in ATC (in BRAF, PIK3CA, CTNNB1 and AXIN1 genes) were rarely detected, undermining their role in ATC development. The study of the microRNA stability in thyroid tumourigenesis is still ongoing. Turnover rates were determined in a dedifferentiated thyroid carcinoma cell line (BCPAP), under standard conditions. A non-disruptive pulse-labelling method was applied and the decrease in microRNA expression along the time was quantified by two-colour arrays. These results represent the first report for an atlas of human microRNA half-lives. The determined average half-life for 249 microRNA was 2.5 days, a stability time more comparable to proteins than to mRNA. The comparison of these data with decay rates in normal thyroid cells will probably uncover miRNA for which stability is deregulated. In conclusion, this project presents several genes and molecular events that were found deregulated in PDTC and ATC. More importantly, these represent potential therapeutic targets that can be used for the treatment of these highly aggressive tumours, in the near future.O cancro da tiróide constitui a neoplasia mais comum do sistema endócrino (van der Zwan et al., 2012), representando nos homens e nas mulheres respectivamente, cerca de 1 e 3% dos casos de cancro, estimados em Portugal (Ferlay et al., 2010a) e no mundo (Ferlay et al., 2010b). Os tumores com origem no epitélio folicular representam 90 a 95% das neoplasias da tiróide (Nikiforov and Nikiforova, 2011), e de acordo com as características morfológicas e clínicas, os tumores foliculares malignos são subdivididos em carcinomas bem-diferenciados (WDTC), carcinomas pouco diferenciados (PDTC) e carcinomas indiferenciados ou anaplásicos (ATC). Os tumores foliculares da tiróide representam assim, um modelo clássico do processo tumoral pelo qual uma célula epitelial possui potencial para originar diferentes tipos de tumores, cada um com características patológicas distintas. Nos WDTC incluem-se os carcinomas papilares (PTC) e os carcinomas foliculares (FTC), sendo os PTC os mais frequentes, representando cerca de 80 a 90% dos casos (Kondo et al., 2006; Nikiforov and Nikiforova, 2011). Os WDTC são, em geral, tratados eficazmente com cirurgia e iodo radioactivo, e cerca de 90% dos doentes com menos de 45 anos ficam curados (DeLellis et al., 2004). Por outro lado, os PDTC e os ATC apresentam escassa diferenciação folicular e comportam-se de forma altamente agressiva. Em particular, os ATC apresentam uma progressão clínica muito rápida, e em geral, manifestam no diagnóstico inicial, uma extensa invasão dos tecidos adjacentes e metástases à distância, nomeadamente pulmonares, ósseas ou cerebrais (Muro-Cacho and Ku, 2000; DeLellis et al., 2004). Apesar dos ATC contribuírem apenas para 1 a 2% dos tumores da tiróide (Kondo et al., 2006; Nikiforov and Nikiforova, 2011), são responsáveis por 14 a 50% das mortes relacionadas com cancro da tiróide. Estão associados a uma sobrevivência média de 3 a 5 meses (DeLellis et al., 2004; Nagaiah et al., 2011), representando assim uma das neoplasias mais letais. Os PDTC apresentam características morfológicas e clínicas intermédias entre os WDTC e ATC (Lam et al., 2000; Volante et al., 2004; Sanders et al., 2007; Nambiar et al., 2011). A taxa média de sobrevida aos 5 anos, dos doentes com PDTC é de cerca de 50% e a mortalidade deve-se sobretudo a metástases pulmonares e ósseas (DeLellis et al., 2004; Nambiar et al., 2011). Os PDTC e em especial, os ATC, são refractários às formas convencionais de tratamento (cirurgia e iodo radioactivo). Na maioria dos casos, a ressecção dos tumores não é viável e a quimio- e a radioterapia demonstram um efeito reduzido na sobrevivência dos doentes (Sanders et al., 2007; Smallridge et al., 2009; Abate and Smallridge, 2011). Assim, é de extrema importância a implementação de novas modalidades terapêuticas que sejam eficazes nestas neoplasias. A transformação do epitélio folicular da tiróide resulta tipicamente de alterações genéticas que envolvem componentes das vias de sinalização MAPK-ERK e PI3K-AKT, essenciais na regulação da proliferação e homeostasia celular. Nos PTC detectam-se, de forma mutuamente exclusiva (Kimura et al., 2003; Nikiforov and Nikiforova, 2011), mutações activantes nos genes que codificam a cinase de serina/treonina BRAF e as GTPases H-, K- e NRAS, sendo a mutação do gene BRAF a alteração mais frequente (Kimura et al., 2003; Fugazzola et al., 2006). Entre os WDTC, as mutações nos genes RAS estão associadas a tumores com morfologia folicular, sendo detectadas na variante folicular dos PTC e em FTC (Vasko et al., 2003; Zhu et al., 2003; Di Cristofaro et al., 2006). As características clínicas e histológicas dos carcinomas da tiróide sugerem uma contínua perda da diferenciação celular (um processo de desdiferenciação) pelo qual ocorre um aumento progressivo da agressividade. Considera-se que os PDTC e ATC possam surgir directamente a partir da célula folicular ou derivarem de tumores pré-existentes. Este último modelo é apoiado pela frequente detecção de áreas bem-diferenciadas em casos de PDTC e ATC (Nikiforova et al., 2003a; DeLellis et al., 2004; Quiros et al., 2005; Takano et al., 2007a; Wang et al., 2007a; Santarpia et al., 2008; Ricarte-Filho et al., 2009). Várias evidências moleculares sugerem, de igual modo, que os WDTC podem progredir para PDTC e para ATC. A detecção dos oncogenes RAS e BRAF em casos de PDTC e ATC (Garcia- Rostan et al., 2003; Smallridge et al., 2009; Volante et al., 2009) e a presença destas mutações em componentes diferenciadas e indiferenciadas de um mesmo tumor (Oyama et al., 1995; Nikiforova et al., 2003a; Begum et al., 2004; Cohen et al., 2004; Takano et al., 2007a; Costa et al., 2008; Schwertheim et al., 2009), sugerem um processo de progressão a partir de WDTC. Além disso, estas mutações podem coexistir com alterações no gene supressor de tumores TP53 (Lam et al., 2000; Quiros et al., 2005; Wang et al., 2007), no gene CTNNB1 que codifica o efector β-catenina da via WNT (Garcia-Rostan et al., 2001) e no PIK3CA que codifica a subunidade catalítica da cinase PI3K da via PI3K-AKT (Lam et al., 2000; Garcia-Rostan et al., 2001; Garcia-Rostan et al., 2005; Quiros et al., 2005; Hou et al., 2007a; Wang et al., 2007a; Liu et al., 2008; Santarpia et al., 2008; Ricarte-Filho et al., 2009). Estas alterações, que são detectadas exclusivamente, ou com maior frequência em PDTC e ATC, demonstram, assim, a acumulação e a cooperação de eventos específicos durante a desdiferenciação. Os microRNA (miRNA), que com frequência se encontram desregulados no processo oncogénico (Calin and Croce, 2006), podem apresentar padrões de expressão anómalos nos tumores da tiróide (Nikiforova et al., 2009). Cada um destes RNA, que são não-codificantes, pode estar envolvido na regulação de centenas de transcriptos-alvo, mediando a sua degradação ou a repressão da translacção proteica (Carthew and Sontheimer, 2009). Assim, os miRNA podem constituir potenciais agentes terapêuticos, na medida em que afectam simultaneamente várias vias de sinalização, impedindo os mecanismos compensatórios das células tumorais (Lujambio and Lowe, 2012). Os níveis de expressão dos miRNA numa célula são o resultado tanto da sua biossíntese como da sua degradação. Enquanto que a via de biossíntese se encontra bem caracterizada (Cullen, 2006), o processo de degradação e o tempo de semi-vida dos miRNA, são relativamente desconhecidos. A determinação da estabilidade e a caracterização dos factores que influenciam a degradação destas moléculas, representam, portanto, áreas importantes para a investigação da potencial utilidade terapêutica dos miRNA. A análise de expressão génica global no campo da oncologia demonstrou ser vantajosa no prognóstico e classificação dos tumores, assim como, na identificação de potenciais alvos terapêuticos (Chung et al., 2002; Nevins and Potti, 2007). Esta metodologia tem sido extensivamente utilizada na tiróide, especialmente no estudo dos diferentes subtipos de WDTC (Griffith et al., 2006; Riesco-Eizaguirre and Santisteban, 2007). No caso dos PDTC e ATC, a análise da expressão génica global em tumores primários encontra-se restrita a poucos estudos. A comparação dos perfis de expressão de ATC e PDTC com WDTC e tecido tiroideu normal (TN) permitiu identificar assinaturas moleculares relacionadas com a proliferação e ciclo celular, instabilidade cromossómica, adesão, motilidade celular e perda da função tiróidea (Salvatore et al., 2007; Montero-Conde et al., 2008; Hébrant et al., 2012). Com o objectivo de elucidar os mecanismos envolvidos na progressão e na agressividade dos PDTC e ATC e identificar novos alvos para o tratamento destes tumores, compararam-se os perfis de expressão génica globais de amostras de TN e de WDTC com PDTC e ATC. Tendo em conta os resultados obtidos, procedeu-se a uma extensa pesquisa de mutações em genes envolvidos em processos celulares desregulados nos PDTC e ATC, e correlacionaramse os dados obtidos com os aspectos clínico-patológicos dos doentes. Por fim, o estudo do turnover de RNA através da marcação com um análogo da uridina (4-tiouridina), foi usado para determinar os tempos de semi-vida de miRNA numa linha celular de carcinoma desdiferenciado da tiróide. A comparação da expressão génica global entre 5 PDTC, 19 WDTC e 3 TN, e entre 5 ATC e 4 TN revelou que os PDTC e ATC exibem, em comum, genes desregulados com expressão aumentada associados ao ciclo celular, proliferação celular, segregação cromossómica e ao “checkpoint” do fuso mitótico, enquanto que, os genes sub-expressos estavam principalmente relacionados com a adesão celular. Os ATC apresentaram como eventos específicos (ausentes na análise dos PDTC), o aumento de expressão de componentes da via do TGF-β e a sub-expressão de genes associados à função e metabolismo tiroideu, à morfologia epitelial e às junções celulares. A análise não-supervisionada da semelhança génica entre as amostras e a correlação com a pesquisa de alterações do RAS e BRAF, demonstrou que as variantes foliculares de PTC são os precursores mais prováveis dos PDTC com mutação no gene RAS. Por outro lado, a comparação dos genes diferencialmente expressos em cada tipo de tumor em relação ao tecido normal, permitiu avaliar que os ATC partilhavam maior número de genes com os PTC clássicos do que com os restantes tumores, o qual suporta a provável origem dos ATC a partir dos PTC clássicos. A análise dos genes diferencialmente expressos entre cada tipo de tumor e o TN revelou que 307 de 494 (60%) dos genes eram sobre-expressos em PTC, enquanto que 137 de 171 (80%) estavam subexpressos em FTC, 92 de 107 (86%) estavam sub-expressos em PDTC e 983 de 1333 (74%) estavam com expressão diminuída nos ATC. Validaram-se por RT-PCR quantitativo em grupos independentes de tumores, o gene UHRF1, associado à proliferação e identificado como sobre-expressos nos PDTC em relação ao tecido normal e o gene ITIH5, relacionado com a adesão celular e sub-expresso nos PDTC em relação ao tecido normal. Adicionalmente, caracterizou-se o gene CDKN3, o qual codifica um inibidor das cinases dependentes de ciclina (CDK) que foi identificado, na análise de expressão, como o gene mais sobre-expresso nos ATC, tendo-se confirmado a sua expressão aumentada em ATC, bem como em PDTC relativamente ao TN. De acordo com a sua função supressora de tumor, observou-se uma expressão significativa de formas de splicing aberrantes apenas nos ATC. Devido ao papel importante que a activação da via do TGF-β apresenta na promoção da transição epitelial-mesenquimal (EMT) (Huber et al., 2005), validou-se a expressão do gene SNAI2, regulador do EMT, e cuja expressão é induzida por esta via (Xu et al., 2009). Confirmou-se assim, a sobre-expressão específica deste gene em ATC. Tendo em conta os resultados obtidos nos estudos de expressão génica global dos PDTC e ATC, procedeu-se a uma extensa análise mutacional em genes envolvidos na regulação do ciclo celular como os inibidores de CDK [CDKN1A (p21CIP1), CDKN1B (p27KIP1), CDKN2A (p14ARF, p16INK4A), CDKN2B (p15INK4B) e CDKN2C (p18INK4C)], genes envolvidos na adesão celular (AXIN1, que codifica um regulador negativo da via WNT) e genes cujo envolvimento nestes tumores fora previamente descrito (H-, K-, NRAS, BRAF, PIK3CA, TP53 e CTNNB1). A pesquisa de alterações patogénicas em 26 ATC e 22 PDTC revelou que as mutações dos genes TP53 (presentes em 42% dos ATC e 27% dos PDTC) e RAS (presentes em 31% dos ATC e 18% dos PDTC) são as mais frequentes nestes tumores. As mutações nestes genes apresentaram mútua exclusividade (P=0,0354) e a sua presença estava associada a um menor tempo de sobrevida global dos doentes com PDTC e ATC (P=0,0383). No caso dos inibidores de CDK identificou-se, pela primeira vez, que alterações nos genes CDKN2A e CDKN2B podem estar envolvidos em cerca de 25% dos PDTC. Por outro lado, as mutações previamente descritas como frequentes em ATC, tais como BRAF, PIK3CA, AXIN1 ou CTNNB1 (Smallridge et al., 2009), foram identificadas em menos de 8% dos casos. O método não-disruptivo de marcação de RNA com 4-tiouridina, foi optimizado e utlizado, pela primeira vez, no estudo da estabilidade dos miRNA. A incubação de uma linha celular de tumor desdiferenciado da tiróide (BCPAP), com 200 μM 4-tiouridina durante 24 horas, revelou não ser tóxico para as células, não afectar a expressão dos miRNA e permitiu marcar, purificar e quantificar os miRNA de forma precisa. Recorrendo a “arrays” de expressão de miRNA, obtiveram-se os tempos de semi-vida para 249 miRNA, cujo tempo médio foi de 2,5 dias, variando desde 22 horas (miR-208a e miR-107) até mais de 5,5 dias (miR-1321 e miR-320d). Estes dados revelam que os miRNA apresentam uma maior estabilidade que os RNA mensageiros (Yang et al., 2003; Friedel et al., 2009), sendo comparável à estabilidade proteica (Boisvert et al., 2012). O nosso estudo encontra-se em desenvolvimento mas, pretende-se identificar os factores determinantes para a degradação de miRNA. Em especial, a comparação destes dados com tempos de semi-vida determinados em células normais da tiróide, permitirá identificar miRNA cuja estabilidade se encontra desregulada no processo oncogénico. Em suma, no presente trabalho identificaram-se várias vias e genes que poderão representar alvos para intervenção terapêutica, com impacto no seguimento clínico e na sobrevivência de doentes com tumores agressivos, como são os PDTC e ATC.Fundação para a Ciência e a Tecnologia (FCT, SFRH/BD/46096/2008); Fundação Calouste Gulbenkian; Chronic Disease Research Centre; Portuguese Society of Endocrinology, Diabetes and Metabolism

Multifunctional Nanocarriers for Combination Cancer Therapy

Combination therapy of multiple drugs with different mechanisms can overcome drug resistance, improve safety and efficacy, and thus becomes a promising strategy in cancer treatment. However, the difference of physiochemical and pharmacokinetic properties of the combined drugs might lead to suboptimal uptake and biodistribution of two drugs at the tumor sites. Multifunctional nanocarriers merge as an ideal strategy to circumvent these limitations and improve the therapeutic effect of anticancer agents. A dual-functional nanocarrier was first developed based on the PEG-conjugated S-trans, trans-farnesylthiosalicylic acid (FTS) for delivery of paclitaxel (PTX). FTS is a potent nontoxic RAS antagonist. To facilitate the release of FTS from the carrier and improve the drug loading capacity and formulation stability, both disulfide linkage and Fluorenylmethyloxycarbonyl (Fmoc) group were incorporated to PEG5k-FTS2 to form a PEG5k-Fmoc-S-S-FTS2 micellar system. PTX-loaded PEG5k-Fmoc-S-S-FTS2 micelles exhibited a superior synergistic therapeutic effect in vitro and in vivo. Despite the potent therapeutic effect, the drug content from PEG-conjugated prodrug nanocarriers is relatively low. In order to solve this problem, drug-based monomer was polymerized to increase the number of drug units in each polymer molecule. Here a PPMP-based prodrug copolymer micelle (POEG15-b-PPPMP7), in which each carrier molecule contains 7 units of PPMP, was developed. PPMP is a potent inhibitor of glucosylceramide synthase (GCS), the major catabolic enzyme of ceramide. Doxorubicin (DOX)/POEG-b-PPPMP micellar system exhibited significantly improved antitumor response in vitro and in vivo. The polymeric prodrug-based carrier strategy was also applied to another drug, suberoylanilide hydroxamic acid (SAHA), and developed a well-characterized POEG15-b-PSAHA12 nanocarrier. SAHA is a histone deacetylase inhibitor (HDACI) approved by FDA. In vivo delivery of DOX via POEG-b-PSAHA led to significant inhibition of 4T1.2 tumor growth. However, not all targets have small molecule drugs. Better synergism would be achieved through the combination of small molecule drugs (for known targets) and gene therapeutics for “undruggable” targets. This inspired us to develop a creatine-based polymer (POEG-PCre) for the co-delivery of nucleic acids and chemodrugs. POEG-PCre carrier is effective in co-delivery of DOX and bioengineered miR-34a to tumor cells and specific accumulation to lung tissue for the treatment of breast cancer lung metastasis

INVESTIGATING THE ROLE OF POST-TRANSLATIONAL MODIFICATIONS IN THE CORE RAS GTPASE DOMAIN

Ras proteins are the most commonly mutated oncoproteins in cancer (~30%). Oncogenic, activating Ras mutations are known drivers of the deadliest human cancers, including lung, pancreatic and colorectal cancers. Ras proteins function as critical regulators of cellular growth by acting as molecular switches, cycling between active, GTP- and inactive, GDP-bound states. In their active form, Ras proteins signal through downstream pathways that regulate cellular growth, differentiation and apoptosis. Early attempts to target Ras proteins (farnesyltransferase inhibitors) were directed toward inhibiting key carboxyl (C)-terminal lipid post-translational modifications (PTMs), which are crucial for proper Ras localization and function at the cellular membrane. Despite their failure, FTIs represent the first direct targeting efforts of Ras proteins. Promising new classes of anti-cancer drugs directed at targeting the dysregulation of PTM status in cancers (kinase inhibitors, histone deacetylase inhibitors, HDACi and methyltransferase inhibitors) have demonstrated multiple clinical successes in recent years. PTMs have been demonstrated to alter protein stability and localization as well as protein-protein interactions in several non-histone cancer-related proteins. While PTMs have been extensively studied in the C-terminus of Ras proteins, their role remains poorly understood in the core Ras guanine nucleotide binding domain (GTPase domain). Monoubiquitylation and acetylation within the core Ras GTPase domain have been demonstrated to modulate Ras protein activity, signaling and tumorigenesis, suggesting that PTMs in this region are capable of regulating Ras behavior. Further, aberrant dysregulation in the balance of PTMs has been characterized in several cancer types, including the Ras-driven pancreatic cancer. It is therefore reasonable that Ras PTMs may present a novel avenue for therapeutic targeting in cancer. Despite more than three decades of research, Ras has remained an elusive target for cancer therapy. We have recently identified novel sites of PTMs in Ras proteins at highly conserved residues within the core GTPase domain. Herein, we present highly innovative and novel methods of generating both acetyl- and methyl-lysine in intact Ras proteins. With the combined use of biochemical, structural, cellular and computational data, we provide mechanistic insight into the regulation Ras proteins by PTMs and also provide rationale for novel therapeutic targeting approaches in Ras-driven cancers.Doctor of Philosoph

Development of a novel antibody drug conjugate for the treatment of pancreatic adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal common cancer in the United States: in 2017 there will be around 54,000 new cases and 43,000 patient deaths. (SEER, 2017) The high mortality of PDAC is related to late disease presentation and aggressiveness; nearly 52% of patients present with metastatic disease at the time of diagnosis. (SEER, 2017) Current treatments have marginal improvements on survival, with the most efficacious treatment, gemcitabine and nab-paclitaxel, having a median survival of 12.2 months. (Wu 2018) Failure of current PDAC treatments is attributed to the inefficacy of systemic chemotherapeutics and the development of resistance. (Rahib, 2014) The Dual Endothelin1/Signal PeptideVEGF receptor (DEspR), represents a promising therapeutic target for the treatment of PDAC: it is a highly expressed, specific tumor antigen, which is involved in tumor vasculogenesis and cancer stem cell (CSC) survival. DEspR is a developmentally crucial receptor, responsible for early angiogenesis and neural crest migration, with minimal expression in normal adult tissue. In vitro and in vivo studies of anti-DEspR therapy in PDAC have shown efficacy in decreasing CSC survival, tumor angiogenesis, and improving overall survival in xenograft models of PDAC, with anti-DEspR therapy being a promising candidate for clinical use. Furthermore, anti-DEspR therapy seems to augment chemotherapeutic therapy in vitro and in vivo, suggesting that a DEspR-targeted antibody drug conjugate (ADC) would be highly effective. ADCs are a re-emerging drug class with significant promise. Initial failures of ADCs in clinic were related to poor antigen specificity and failures in drug conjugation chemistry to minimally impact the antibody. To develop our ADC, I have developed a novel method of site-specific conjugation that relies on a novel method of supramolecular assembly. My system employs two specific protein sequences that do not self-interact, and tightly assemble through coulombic and hydrophobic interactions, allowing site-specific, stoichiometric self-assembly. To facilitate stable drug delivery, I have synthesized a novel enzymatically cleavable tyrosine-clickable linker, which prevents drug release prior to tumor delivery. Both further investigation into the efficacy of anti-DEspR therapy, and the development of a stoichiometric, site-specific, stable method for drug loading will provide an advancement in anti-cancer therapy

Cancer Nanomedicine

This special issue brings together cutting edge research and insightful commentary on the currentl state of the Cancer Nanomedicine field

Dichotomic role of NAADP/two-pore channel 2/Ca2+ signaling in regulating neural differentiation of mouse embryonic stem cells

Poster Presentation - Stem Cells and Pluripotency: abstract no. 1866The mobilization of intracellular Ca2+stores is involved in diverse cellular functions, including cell proliferation and differentiation. At least three endogenous Ca2+mobilizing messengers have been identified, including inositol trisphosphate (IP3), cyclic adenosine diphosphoribose (cADPR), and nicotinic adenine acid dinucleotide phosphate (NAADP). Similar to IP3, NAADP can mobilize calcium release in a wide variety of cell types and species, from plants to animals. Moreover, it has been previously shown that NAADP but not IP3-mediated Ca2+increases can potently induce neuronal differentiation in PC12 cells. Recently, two pore channels (TPCs) have been identified as a novel family of NAADP-gated calcium release channels in endolysosome. Therefore, it is of great interest to examine the role of TPC2 in the neural differentiation of mouse ES cells. We found that the expression of TPC2 is markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebound during the late stages of neurogenesis. Correspondingly, perturbing the NAADP signaling by TPC2 knockdown accelerates mouse ES cell differentiation into neural progenitors but inhibits these neural progenitors from committing to the final neural lineage. Interestingly, TPC2 knockdown has no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Overexpression of TPC2, on the other hand, inhibits mouse ES cell from entering the neural lineage. Taken together, our data indicate that the NAADP/TPC2-mediated Ca2+signaling pathway plays a temporal and dichotomic role in modulating the neural lineage entry of ES cells; in that NAADP signaling antagonizes ES cell entry to early neural progenitors, but promotes late neural differentiation.postprin

Farnesylthiosalicylic acid sensitizes hepatocarcinoma cells to artemisinin derivatives

<div><p>Dihydroartemisinin (DHA) and artesunate (ARS), two artemisinin derivatives, have efficacious anticancer activities against human hepatocarcinoma (HCC) cells. This study aims to study the anticancer action of the combination treatment of DHA/ARS and farnesylthiosalicylic acid (FTS), a Ras inhibitor, in HCC cells (Huh-7 and HepG2 cell lines). FTS pretreatment significantly enhanced DHA/ARS-induced phosphatidylserine (PS) externalization, Bak/Bax activation, mitochondrial membrane depolarization, cytochrome <i>c</i> release, and caspase-8 and -9 activations, characteristics of the extrinsic and intrinsic apoptosis. Pretreatment with Z-IETD-FMK (caspase-8 inhibitor) potently prevented the cytotoxicity of the combination treatment of DHA/ARS and FTS, and pretreatment with Z-VAD-FMK (pan-caspase inhibitor) significantly inhibited the loss of ΔΨm induced by DHA/ARS treatment or the combination treatment of DHA/ARS and FTS in HCC cells. Furthermore, silencing Bak/Bax modestly but significantly inhibited the cytotoxicity of the combination treatment of DHA/ARS and FTS. Interestingly, pretreatment with an antioxidant N-Acetyle-Cysteine (NAC) significantly prevented the cytotoxicity of the combination treatment of DHA and FTS instead of the combination treatment of ARS and FTS, suggesting that reactive oxygen species (ROS) played a key role in the anticancer action of the combination treatment of DHA and FTS. Similar to FTS, DHA/ARS also significantly prevented Ras activation. Collectively, our data demonstrate that FTS potently sensitizes Huh-7 and HepG2 cells to artemisinin derivatives via accelerating the extrinsic and intrinsic apoptotic pathways.</p></div