CD133 and ALDH1 as Prospective Markers for Cancer Stem Cells in Ileal Carcinoids

Roten till cancer? Cancer är en ledande orsak till en stor mängd dödsfall i världen och därför en fruktad sjukdom. Forskningen gör dock framsteg och man kommer allt närmre svaren på gåtorna. På senare år har det föreslagits att en speciell typ av celler, cancerstamceller, är drivande och ligger bakom flera av cancersjukdomarnas egenskaper. Alla organismer är uppbyggda av celler, den stora majoriteten med en egen arvsmassa – DNA. Ibland går det fel när en av cellerna ska dela sig och en förändring i arvsmassan, en mutation, uppkommer. Dessa mutationer ackumuleras över tid och har man otur kan vissa mutationer i kombination orsaka att cellen övergår till att bli en snabbt och okontrollerbart delande cancercell. Kan man hitta och isolera dessa cancerstamceller hoppas man kunna hitta de egenskaper som skiljer dem ifrån friska celler och på så vis skapa läkemedel som slår emot just dessa. Att leta efter cancerstamceller I detta arbete har jag försökt isolera och karaktärisera cancerstamceller som härstammar ifrån carcinoider, en typ av cancertumörer, i tunntarmen. För att åstadkomma detta användes sju så kallade markörer, molekyler som andra forskare tror uttrycks extra mycket i stamceller. För att se om dessa molekyler uttrycks i cellerna färgades de in med antikroppar. För varje markör användes en specifik antikropp som band till just denna och gav ifrån sig ett sken. Skenet studerades sedan i en flödescytometer med sorteringsfunktion, där celler som var intressanta kunde isoleras. I mitt fall fanns en grupp celler som i högre mån än de andra uttryckte markörerna CD133 och ALDH1. Dessa valdes därför ut för fortsatta experiment. Experimenten bestod i att dels odla cellerna i olika medium för att studera dess egenskaper, men framförallt i att analysera cellernas mRNA. Allt som produceras av en cell har sitt ursprung i DNA, cellens bibliotek. När en molekyl ska produceras kommer DNA-molekylen att läsas av och en kopia av den del som kodar för molekylen, även kallad gen, skapas. Denna kopia består av mRNA och man kan på så vis få reda på hur mycket som produceras av en molekyl genom att se hur mycket mRNA associerat med just den molekylen som finns. Jag studerade 21 olika typer av mRNA associerade med var sin molekyl som i forskning tidigare antytts vara karaktäristiska för just cancerstamceller. De tidigare utsorterade cellerna jämfördes med celler som inte genomgått någon särskild sortering och med celler som inte uttryckte de båda markörerna. Signifikanta skillnader mellan grupperna kunde ses för flera av markörerna. Särskilt de celler som uttryckte högre nivåer av ALDH1 tycks också uttrycka stor mängd av flera andra markörer som tros vara viktiga för cancerstamceller. Handledare: Yvonne Arvidsson Examensarbete - cell- och molekylärbiologi, 30 högskolepoäng, 2011 Forskningsgrupp Ola Nilsson, Sahlgrenska Cancercentrum, Göteborg.In recent years increasing evidence supporting the stem cell hypothesis have emerged and cancer stem cells (CSCs) have been reported to have been isolated in a wide variety of cancers. This CSC population has been attributed to contribute to the therapeutic resistance of many cancers and it is thus of great interest to find new therapeutic options targeting these cells. In ileal carcinoids however not much is known regarding the existence and properties of CSCs. The aim of this study was to identify, isolate and characterize cancer cell populations of ileal carcinoids sorted with fluorescent-activated cell sorting (FACS) using putative cancer stem cell markers. The FACS markers included the six cell surface receptors CD26, CD44, CD56, CD117, CD133 and CD166 as well as an intracellular assay indicating the activity of the enzyme aldehyde dehydrogenase 1 (ALDH1). The CD133 and ALDH1 markers did respectively in both primary patient cultures and a cell line help to identify distinct cell subpopulations which when cultured on low-attachment plastic ware gave rise to sphere-like formations. Analysis of the populations by quantitative real-time PCR (qRT-PCR) showed significant differences in the expression of mRNA relevant to CSC

Extreme Female Promiscuity in a Non-Social Invertebrate Species

Background: While males usually benefit from as many matings as possible, females often evolve various methods of resistance to matings. The prevalent explanation for this is that the cost of additional matings exceeds the benefits of receiving sperm from a large number of males. Here we demonstrate, however, a strongly deviating pattern of polyandry. Methodology/Principal Findings: We analysed paternity in the marine snail Littorina saxatilis by genotyping large clutches (53–79) of offspring from four females sampled in their natural habitats. We found evidence of extreme promiscuity with 15–23 males having sired the offspring of each female within the same mating period. Conclusions/Significance: Such a high level of promiscuity has previously only been observed in a few species of social insects. We argue that genetic bet-hedging (as has been suggested earlier) is unlikely to explain such extreme polyandry. Instead we propose that these high levels are examples of convenience polyandry: females accept high numbers of mating

SMAD4 haploinsufficiency in small intestinal neuroendocrine tumors

Background: Patients with small intestinal neuroendocrine tumors (SINETs) frequently present with lymph node and liver metastases at the time of diagnosis, but the molecular changes that lead to the progression of these tumors are largely unknown. Sequencing studies have only identified recurrent point mutations at low frequencies with CDKN1B being the most common harboring heterozygous mutations in less than 10% of all tumors. Although SINETs are genetically stable tumors with a low frequency of point mutations and indels, they often harbor recurrent hemizygous copy number alterations (CNAs) yet the functional implications of these CNA are unclear. Methods: Utilizing comparative genomic hybridization (CGH) arrays we analyzed the CNA profile of 131 SINETs from 117 patients. Two tumor suppressor genes and corresponding proteins i.e. SMAD4, and CDKN1B, were further characterized using a tissue microarray (TMA) with 846 SINETs. Immunohistochemistry (IHC) was used to quantify protein expression in TMA samples and this was correlated with chromosome number evaluated with fluorescent in-situ hybridization (FISH). Intestinal tissue from a Smad4+/− mouse model was used to detect entero-endocrine cell hyperplasia with IHC. Results: Analyzing the CGH arrays we found loss of chromosome 18q and SMAD4 in 71% of SINETs and that focal loss of chromosome 12 affecting the CDKN1B was present in 9.4% of SINETs. No homozygous loss of chromosome 18 was detected. Hemizygous loss of SMAD4, but not CDKN1B, significantly correlated with reduced protein levels but hemizygous loss of SMAD4 did not induce entero-endocrine cell hyperplasia in the Smad4+/− mouse model. In addition, patients with low SMAD4 protein expression in primary tumors more often presented with metastatic disease. Conclusions: Hemizygous loss of chromosome 18q and the SMAD4 gene is the most common genetic event in SINETs and our results suggests that this could influence SMAD4 protein expression and spread of metastases. Although SMAD4 haploinsufficiency alone did not induce tumor initiation, loss of chromosome 18 could represent an evolutionary advantage in SINETs explaining the high prevalence of this aberration. Functional consequences of reduced SMAD4 protein levels could hypothetically be a potential mechanism as to why loss of chromosome 18 appears to be clonally selected in SINETs

177 Lu-octreotate therapy for neuroendocrine tumours is enhanced by Hsp90 inhibition

Lu-177-octreotate is an FDA-approved radionuclide therapy for patients with gastroenteropancreatic neuroendocrine tumours (NETs) expressing somatostatin receptors. The Lu-177-octreotate therapy has shown promising results in clinical trials by prolonging progression-free survival, but complete responses are still uncommon. The aim of this study was to improve the Lu-177-octreotate therapy by means of combination therapy. To identify radiosensitising inhibitors, two cell lines, GOT1 and P-STS, derived from small intestinal neuroendocrine tumours (SINETs), were screened with 1224 inhibitors alone or in combination with external radiation. The screening revealed that inhibitors of Hsp90 can potentiate the tumour cell-killing effect of radiation in a synergistic fashion (GOT1; false discovery rate < 3.2 x 10(-11)). The potential for Hsp90 inhibitor ganetespib to enhance the anti-tumour effect of Lu-177-octreotate in an in vivo setting was studied in the somatostatin receptor-expressing GOT1 xenograft model. The combination led to a larger decrease in tumour volume relative to monotherapies and the tumour-reducing effect was shown to be synergistic. Using patient-derived tumour cells from eight metastatic SINETs, we could show that ganetespib enhanced the effect of Lu-177-octreotate therapy for all investigated patient tumours. Levels of Hsp90 protein expression were evaluated in 767 SINETs from 379 patients. We found that Hsp90 expression was upregulated in tumour cells relative to tumour stroma in the vast majority of SINETs. We conclude that Hsp90 inhibitors enhance the tumour-killing effect of Lu-177-octreotate therapy synergistically in SINET tumour models and suggest that this potentially promising combination should be further evaluated

The neuroendocrine phenotype, genomic profile and therapeutic sensitivity of GEPNET cell lines

Experimental models of neuroendocrine tumour disease are scarce, and no comprehensive characterisation of existing gastroenteropancreatic neuroendocrine tumour (GEPNET) cell lines has been reported. In this study, we aimed to define the molecular characteristics and therapeutic sensitivity of these cell lines. We therefore performed immunophenotyping, copy number profiling, whole-exome sequencing and a large-scale inhibitor screening of seven GEPNET cell lines. Four cell lines, GOT1, P-STS, BON-1 and QGP-1, displayed a neuroendocrine phenotype while three others, KRJ-I, L-STS and H-STS, did not. Instead, these three cell lines were identified as lymphoblastoid. Characterisation of remaining authentic GEPNET cell lines by copy number rofiling showed that GOT1, among other chromosomal alterations, harboured losses on chromosome 18 encompassing the SMAD4 gene, while P-STS had a loss on 11q. BON-1 had a homozygous loss of CDKN2A and CDKN2B, and QGP-1 harboured amplifications of MDM2 and HMGA2. Whole-exome sequencing revealed both disease-characteristic mutations (e.g. ATRX mutation in QGP-1) and, for patient tumours, rare genetic events (e.g. TP53 mutation in P-STS, BON-1 and QGP-1). A large-scale inhibitor screening showed that cell lines from pancreatic NETs to a greater extent, when compared to small intestinal NETs, were sensitive to inhibitors of MEK. Similarly, neuroendocrine NET cells originating from the small intestine were considerably more sensitive to a group of HDAC inhibitors. Taken together, our results provide a comprehensive characterisation of GEPNET cell lines, demonstrate their relevance as neuroendocrine tumour models and explore their therapeutic sensitivity to a broad range of inhibitors

Small intestinal neuroendocrine tumours - Disease models, tumour development, and remedy

Small intestinal neuroendocrine tumours (SINETs) are malignant neoplasms which at the time of diagnosis often present with distant metastasis. The field of SINET research faces several challenges. There is a lack of preclinical models for studying SINETs, and it is unclear how well currently available models actually recapitulate the tumour disease. The genetic changes that underlie SINET tumour development are largely unknown and, lastly, curative therapy is rarely achieved. Novel therapies, such as the recently FDA-approved 177Lu-octreotate therapy and up-and-coming immunotherapies need to be further investigated to deliver better response rates for SINET patients. In our first two papers (papers I and II), we sought to evaluate frequently used and readily available gastroenteropancreatic neuroendocrine tumour (GEPNET) cell lines as models of neuroendocrine tumour disease. We investigated the characteristics of these cell lines in terms of their neuroendocrine phenotype, genomic background, and therapeutic sensitivity. While several cell lines exhibited an expected neuroendocrine differentiation and harboured genetic alterations characteristic of the GEPNET disease, three cell lines did not. In fact, it turned out that one of the most frequently used cell lines in the field – KRJ-I, together with the cell lines L-STS and H-STS, were incorrectly identified and instead lymphoblastoid cell lines (EBV-immortalised B-lymphocytes). This might have led to the incorrect use and potentially faulty conclusions in a number of GEPNET studies. Among authentic cell lines, we performed a large-scale inhibitor sensitivity screening and predicted that SINETs would be more sensitive to HDACi compared to pancreatic neuroendocrine tumours (PanNET) and PanNET more sensitive to MEKi compared to SINET. The prediction was supported by subsequent experiments with primary tumour cells. In our third paper (paper III), we evaluated a mechanism by which hemizygous loss of SMAD4 could lead to SINET initiation and/or progression by acting as a haploinsufficient tumour suppressor. We found that loss of SMAD4 was associated with a decrease in corresponding mRNA and protein, and that this correlated to patient survival. We also found that the amount of SMAD4 protein in the primary tumour could predict whether the patient presented with distant metastasis. In our last papers (papers IV and V), we investigated the potential for two novel treatment strategies for SINETs. In paper IV we identified an inhibitor, the heat shock protein 90 inhibitor ganetespib, that could synergistically enhance the 177Lu-octreotate therapy for SINETs. Ganetespib was initially found to sensitise SINETs to radiation in a large-scale inhibitor synergy screening, and its radiosensitising effect for radionuclide treatment of SINETs was validated both in mouse xenografts and in primary patient tumours. Lastly, in paper V we characterised the SINET immune microenvironment. Using immunohistochemistry and flow-cytometry we detailed the immune cell composition of the SINET immune microenvironment and could demonstrate the successful isolation and expansion of tumour-infiltrating lymphocytes. We saw that after infiltrating lymphocytes were expanded they could degranulate when challenged with autologous tumour cells. In conclusion, these studies have provided a thorough characterisation of authentic, and provided important information regarding misidentified, frequently used gastroenteropancreatic cell lines. It has also investigated the role of hemizygous SMAD4 loss in the development of SINETs and demonstrated the potential of two novel therapies for SINETs: 177Lu-octreotate combined with Hsp90i ganetespib and immunotherapy

Expression profiling of small intestinal neuroendocrine tumors identifies subgroups with clinical relevance, prognostic markers and therapeutic targets.

We wanted to define the transcriptome of small intestinal neuroendocrine tumors in order to identify clinically relevant subgroups of tumors, prognostic markers and novel targets for treatment. Genome-wide expression profiling was conducted on tumor biopsies from 33 patients with well-differentiated neuroendocrine tumors of the distal ileum and metastatic disease at the time of diagnosis. Unsupervised hierarchical clustering analysis identified three groups of tumors. The largest group, comprising half of the tumors, was characterized by longer patient survival and higher expression of neuroendocrine markers, including SSTR2. Tumors with higher grade (G2/3) or gain of chromosome 14 were associated with shorter patient survival and increased expression of cell cycle-promoting genes. Pathway analysis predicted the prostaglandin E receptor 2 (PTGER2) as the most significantly activated regulator in tumors of higher grade, whereas Forkhead box M1 (FOXM1) was the most significantly activated regulator in tumors with gain of chromosome 14. Druggable genes identified from expression profiles included clinically proven SSTR2 and also novel targets, for example, receptor tyrosine kinases (RET, FGFR1/3, PDGFRB and FLT1), epigenetic regulators, molecular chaperones and signal transduction molecules. Evaluation of candidate drug targets on neuroendocrine tumors cells (GOT1) showed significant inhibition of tumor cell growth after treatment with tyrosine kinase inhibitors or inhibitors of HDAC, HSP90 and AKT. In conclusion, we have defined the transcriptome of small intestinal neuroendocrine tumors and identified novel subgroups with clinical relevance. We found specific gene expression patterns associated with tumor grade and chromosomal alterations. Our data also suggest novel prognostic biomarkers and therapies for these patients

The number of sires in four half-sib families of <i>Littorina saxatilis</i>.

<p>Four females and their offspring were genotyped at five microsatellite DNA loci. The most likely number was estimated using the likelihood-based software COLONY and the minimum number was calculated using MINSIRES.</p

Full-sib family size distribution in four broods of <i>Littorina saxatilis</i>.

<p>Observed number of offspring per male is estimated in COLONY. Expected number of sires is approximated by a truncated Poisson distribution.</p