Rozprawy doktorskie na temat „Metastasis”

The complex PI3K/mTOR pathway regulates tumor progression via effects on cellular proliferation, apoptosis, autophagy, and motility. New drugs that inhibit the catalytic site of both PI3K and mTOR have shown promise in clinical trials. Here, we report the first use of a novel, dual PI3K/mTOR catalytic site inhibitor (PF-04691502, PF1502) in a xenograft model of breast cancer metastasis to bone. Metastatic MDA-MB-1833 cells showed PI3K/mTOR activation relative to parental MDA-MB-231. Low-dose PF1502 significantly impaired tumor cell motility and invasion in vitro without causing cell cycle arrest, apoptosis, or reduced proliferation. Pre-treatment of tumor cells at this dose reduced bone metastatic outgrowth in vivo. The atypical tumor suppressor, p27KIP1, is phosphorylated in its C-terminal region by multiple AGC kinases downstream of PI3K/mTOR. These phosphorylation events promote cytoplasmic mislocalzation of p27 which, in turn, facilitates inhibition of the RhoA cytoskeletal regulatory protein. The resulting turnover of the actin cytoskeleton is thought to underlie the increased cellular motility attributed to cytoplasmic p27. In MDA-MB-1833 cells, PI3K/mTOR inhibition reduced p27 C-terminal phosphorylation at T157 and T198 and reduced cytoplasmic p27 levels. Overexpression of a p27T157D/T198D phospho-mimetic mutant conferred resistance to the anti-motility effects of PF1502 in vitro. MDA-MB-1833 cells demonstrate p27-dependent inhibition of RhoA-ROCK signaling, as well as p27-dependent motility and invasion in vitro, however, RhoA knockdown did not confer resistance to the anti-motility effects of PF1502. p27shRNA dramatically impaired the bone metastatic outgrowth of MDA-MB-1833 in vivo. In an effort to explore potentially novel RhoA-independent mechanisms whereby cytoplasmic p27 might drive tumor cell motility and metastasis, we turned to the process known as epithelial-to-mesenchymal transition (EMT). The EMT program has been implicated as a critical driver of tumor metastasis in a variety of cancer models. PI3K/mTOR inhibition and shRNA p27 treatment both reversed expression of EMT markers in MDA-MB-1833. Thus, PI3K/mTOR appears to drive p27-dependent motility and metastasis at least in part by induction of an EMT-like phenotype, a novel mechanism through which p27 might act to promote tumor progression. These results provide an important new clinical rationale supporting the use of PI3K/mTOR inhibitors as anticancer agents via their inhibition of tumor invasion and metastasis.

Metastatic prostate cancer is currently incurable. Metastasis is thought to result from changes in the expression of specific metastasis-driving genes, leading to a cascade of activated downstream genes setting the metastatic process in motion. As such, metastasis-driving genes could provide effective therapeutic targets and prognostic biomarkers for improved disease management. In search of potential metastasis-driving genes, genes with elevated expression in patient-derived metastatic LTL-313H prostate cancer tissues, as distinct from non-metastatic LTL-313B tissues, were identified. Among these genes, TIMELESS and DLX1 were promising. Unfortunately, their silencing and overexpression in prostate cancer cells did not lead to inhibition of metastatic properties, indicating that they were not metastasis-driving genes. A different, novel approach was used based on the notion that metastasis-driving genes can activate genes in an amplification cascade fashion. Accordingly, I used the IPA’s Upstream Regulator Analysis tool to analyze the differential gene expression profile of the metastatic and non-metastatic tissues to predict the upstream master regulatory (metastasis-driving) genes accountable for the differential expression. Six candidate genes were identified, including GATA2, a pioneer factor-encoding gene. Elevated GATA2 expression in clinical metastatic prostate cancer specimens correlated with poor patient prognosis. Furthermore, GATA2 gene silencing in human prostate cancer LNCaP cells led to marked reduction in cell proliferation, cell migration, tissue invasion, focal adhesion disassembly and a dramatic change in transcriptional activity, indicating that GATA2 plays a critical role in prostate cancer metastasis. As such, GATA2 could represent a metastasis-driving gene and a potential therapeutic target for inhibiting the growth and metastasis development in prostate cancer. Further analysis of GATA2-regulated genes led to the development of a GATA2-based metastatic gene signature. Its prognostic value was confirmed using two prostate cancer patient cohorts. In addition, it was shown to be a prognostic factor for risk assessment of metastasis development, independent of the widely used D’Amico prognostic classification system. However, a thorough validation is critical and, if successful, the GATA2-based gene signature could lead to a paradigm shift in the management of early prostate cancer. In conclusion, the findings of this study appear to be potentially useful for improved management of metastatic prostate cancer.
Medicine, Faculty of
Graduate

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers with an overall 5-year survival rate < 5%, a rate that has not improved for a long time. The dismal prognosis for PDAC is part due to late detection, often at advanced stages of the disease where patients have developed distant metastases, but also due to chemotherapeutical resistance. Patients eligible for surgical resection of the pancreatic tumour has the best prognosis, but even when resection is successful, patients often relapse with distant metastases within 2 years after surgery. Macrophages promote tumourigenesis and enhance metastasis in many cancer types; however, the role of macrophages in PDAC metastasis is poorly understood. Using an experimental mouse model of liver metastasis, we find that PDAC liver metastasis critically depends on the early recruitment of granulin-secreting metastasis-associated macrophages (MAMs) to the liver that promote metastatic colonisation. Mechanistically, we demonstrate that granulin secretion by MAMs activates resident hepatic stellate cells (HSTCs) into myofibroblasts that secrete extracellular matrix components, including periostin, resulting in a fibrotic microenvironment that sustains metastatic tumour growth. Disruption of MAM recruitment in PI(3)Kγ-deficient mice, chemical depletion of MAMs from established lesions or genetic ablation of granulin reduces HSTC activation and liver metastasis. Adjuvant CTX is standard for patients after surgical resection to eliminate any residual cancer cells, and it improves survival for patients after resection. We find that 45% of mice with metastatic lesions respond to gemcitabine treatment with a reduction in metastatic burden. The metastatic lesions in these mice are characterised by a reduction in αSMA+ myofibroblasts. HSTCs do not seem to promote cancer cell survival in the presence of chemotherapy, but rather constitutes a protective niche that promotes relapse by promoting the growth of pancreatic cancer cells after gemcitabine treatment.

Colorectal cancer is one of the most frequently occurring malignancies and a major cause of cancer death. Distant metastases in this disease most commonly develop in the liver and are often untreatable. Here, we use proteomics to characterise, qualitatively and quantitatively, extracellular matrix (ECM) from colorectal cancer liver metastases. We show that citrullination of the ECM by cancer cell derived peptidyl arginine deiminase 4 (PAD4) is important for the growth of liver metastases. Citrullination of proteins, a posttranslational conversion of arginine residues to citrulline, is well recognised in rheumatoid arthritis, but largely undocumented in cancer. PAD4, a key enzyme responsible for catalysing citrullination, is produced by metastatic colorectal cancer cells and found at higher levels in human liver metastases than in normal liver. Functional significance for citrullination in metastatic growth was evident in murine models where inhibition of citrullination, either globally by pharmacologic inhibition of PADs or specifically in colorectal cancer cells by PAD4 knockdown reduced liver metastatic burden by 3- to 4-fold (P < 0.05). Additionally, citrullination of key extracellular matrix (ECM) component collagen type I in vitro led to greater adhesion and 25-30% decreased migration of colorectal cancer cells (P < 0.05) along with increased expression of characteristic epithelial markers, indicating a role for citrullination in promoting mesenchymal-to-epithelial transition (MET). Overall, our study revealed PAD4- dependent citrullination of the ECM altering mesenchymal-epithelial plasticity in colorectal cancer cells and the progression of liver metastasis. These data indicate that inhibition of citrullination could be exploited to potentially prevent the development of liver metastases in colorectal cancer.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2009.
Includes bibliographical references.
Nearly 90% of cancer mortality from solid tumors is due to metastasis of malignant cells to the distant vital organs. It is now well established that a plethora of stromal cells are present within the tumor, and contribute in various ways to tumor initiation and progression, and plasma membrane proteins are the mediators for tumor-stromal communications. In this thesis, I focused on plasma membrane proteins that may contribute to tumor metastasis. I applied quantitative mass spectrometry technology to first identify plasma proteins that are expressed at different levels in melanoma cells with high versus low metastatic abilities. Using SILAC (stable isotope labeling with amino acids in culture) coupled with nano-spray tandem mass spectrometry, this work led to the discovery of C̲ub Ḏomain C̲ontaining Protein 1 (CDCP1) as one of those differentially expressed transmembrane proteins. We found that CDCP1 is not only a surface marker for cells with higher metastatic potential, it is also functionally engaged in enhancing tumor metastasis. When searching for the underlying mechanisms, we found that CDCP1 is important for soft agar colony-forming abilities, suggesting that CDCP1 might regulate the balance between cell proliferation and anoikis. Making use of 3D Matrigel culture system, we found that CDCP1 also regulates scattered growth of melanoma cells. We speculate these two factors may contribute to enhanced-metastatic ability observed in mice.
(cont.) When investigating signaling pathways that may mediate the functions of CDCP1, we found that overexpression of CDCP1 correlates with hyper-activation of Src family kinases. While wild-type CDCP1 enhances SFK activation, point mutation that abolished CDCP1 functions (in scattered growth and in metastasis) also abolished SFK hyper-activation, suggesting that CDCP1 might function through the activation of SFKs. Such notion was further supported since pharmacological reagents PP2 and Dasatinib, which are two SFK inhibitors, blocked in vitro functions of CDCP1 in scattered growth. Thus the work in this thesis has identified a novel metastasis enhancer, CDCP1, and has gained insight into the mechanisms by which CDCP1 functions.
by Hui Liu.
Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2008.
Includes bibliographical references.
The epithelial cell adhesion molecule E-cadherin is often downregulated during carcinoma progression and metastatic spread of tumors. However, the precise mechanism and molecular basis of metastasis promotion by E-cadherin loss is not completely understood. To investigate its role in metastasis, I utilized two distinct methods of E-cadherin inhibition that distinguish between E-cadherin's cell-cell adhesion and intracellular signaling functions. While the disruption of cell-cell contacts alone does not enable metastasis in vivo, the loss of E-cadherin protein does, through induction of an epithelial-to-mesenchymal transition (EMT), invasiveness and anoikis-resistance. E-cadherin binding partner f3-catenin is necessary but not sufficient for these phenotypes. In addition, gene expression analysis shows that E-cadherin loss results in the induction of multiple transcription factors, at least one of which, Twist, is necessary for E-cadherin loss-induced metastasis. These findings indicate that E-cadherin loss in tumors contributes to metastatic dissemination by inducing wide-ranging transcriptional and functional changes. In addition to promoting metastasis, loss of E-cadherin and the accompanying EMT renders cells resistant to conventional chemotherapeutic drugs. As the cells that have undergone an EMT represent the pool of cancer cells most competent to metastasize and lead to tumor recurrence, it is of vital importance to find therapies that effectively target such cells. Paired cell lines that differ in their differentiation state were utilized to discover compounds with selective toxicity against cells that have undergone an EMT. High-throughput screening of small molecule libraries resulted in a number of compounds that specifically affect the viability of cells that have undergone an EMT while having minimal cytotoxic effects on control epithelial cells. These studies establish a proof-of-principle for discovering compounds that target highly metastatic and otherwise chemotherapy resistant cancer cells.
by Tamer T. Onder.
Ph.D.

En païssos industrialitzats, el càncer de pròstata (CP) és la neoplasia més comunment diagnosticada en homes i la segona causa de mort relacionada amb càncer, donat que els nivells de mortalitat en aquesta població són molt més baixos que els que es troben en els païssos en desenvolupament, es veu un clar benefici en els avenços tant en el diagnòstic precoç com el desenvolupament de teràpies eficients. No obstant, la disseminació metastàtica més que el tumor primari en sí és la responsable dels problemes de mortalitat i morbiditat associats al CP. Les metàstasis esquelètiques estan presents en més d’un 70% dels casos de CP avançat i confereixen alts nivells de morbiditat, un 25% de supervivència als 5 anys i una mitjana de supervivència de 40 mesos després de ser diagnosticats. Tot i que les fractures patològiques i la compressió de la columna vertebral són les complicacions més probables pel pacient metastàtic, el major símptoma és el dolor. Les metàstasis òssies del CP comporten un estat d’acceleració de la remodelació òssia que es caracteritza per una activació patològica tant dels osteoblasts com dels osteoclasts. Aquesta elevada activació dels osteoclasts està directament correlacionada amb un increment en la incidència de les complicacions òssies, de la progressió tumoral i la mort. A més, una vegada el tumor metastatitza a os, la malaltia esdevé incurable i les teràpies actuals són solament pal·liatives i principalment es dirigeixen a les cèl·lules tumorals o als osteoclasts. Per tant, per entendre millor la biologia de les metàstasis òssies del PC i poder investigar noves teràpies és important desenvolupar nous models animals. En aquesta tesi, s’han establert nous models experimentals de metàstasis del CP mitjançant la inoculació de cèl·lules humanes en ratolins immunodeficients per diferents vies, intraòssia, intracardíaca o intratibial. Finalment, diferents estratègies s’han dut a terme per descriure noves dianes moleculars involucrades en el mecanisme de les metàstasis i poder desenvolupar un model adequat per la evaluació de possibles compostos candidats a ser futures aproximacions terapèutiques. Per concloure, aquests models proporcionen una fiable reproducció de la situació clínica i permeten tant la caracterització com el diseny de tractaments efectibles per compendre millor els mecanismes moleculars de les metàstasis òssies del CP.
In industrialized countries, prostate cancer (PCa) is the most common malignancy in men, but mortality rates are much lower than those recorded in developing countries, reflecting benefits from advances in early diagnosis and effective treatment. However, the metastatic disease rather than the primary tumor is responsible for much of the resulting morbidity and mortality. Skeletal metastases occur in more than 70% of cases of late-stage of PCa and they confer a high level of morbidity, a 5 year survival rate of 25% and median survival of approximately 40 months. Though fractures and spinal cord compression are potential complications, the most common symptom of bone metastases is pain. Bone metastases from PCa lead to an accelerated bone turnover state that features pathological activation of both osteoblasts and osteoclasts. Raised activation of osteoclasts is directly correlated with an increased incidence of skeletal complications, cancer progression and death. Further, once tumor metastasizes to bone, the metastatic disease become incurable and current therapies are palliative and mostly target either tumor cells or osteoclasts. Thus, to better understand the biology of PCa bone metastasis and to investigate new therapy options it is crucial to develop new animal models. In this thesis, we have established new experimental models of PCa bone metastasis by intraosseous (i.o.), intracardiac (i.c.) or intratibial (i.t.) inoculation of human PCa cells in immunodeficient mice. Extensive bone metastasis were monitored by in vivo bioluminescence imaging. Different strategies were performed to describe new molecular targets involved in the mechanisms of PCa bone metastasis and to make a suitable model for evaluating novel compounds as future therapeutic approaches. To conclude, these models provide a reliable reproduction of the clinical situation and allows characterization and design effective treatments by better understanding the molecular mechanisms of PCa bone metastasis.

The identification of genes that mediate metastasis is pivotal to better understand the mechanism, to develop novel drugs and to stratify patients with highest risk and consecutively administrate them preventive treatments. Despite significant advances on knowledge, diagnosis and treatment of cancer, metastasis remains the major cause of cancer-associated deaths. Bone is one of the most common organs affected by metastatic lesions for its permeability and favorable conditions for cellular growth. Constant remodeling in bone homeostasis implies an incessant degradation of the bone that release high concentrations of growth factors into the microenvironment. Thereby, growth factors benefit both, formation of new bone and/or tumor cell growth. Although the importance of bone metastatic lesions in cancer patients and the advances on the knowledge of this process, few treatments are currently administrated to patients that suffer this disease, specifically Denosumab and Zoledronic acid (ZOL). Importantly, these treatments can improve the symptoms of bone lesions but cannot cure or reverse metastasis. This fact reflects the need to detect and tackle new targetable elements to reduce bone metastatic lesions. Many molecular mechanisms have been described in bone metastasis, but only one predictor gene has been identified, MAF. MAF is a transcription factor that has been previously involved in carcinogenesis, specially in multiple myeloma (MM) and human angioimmunoblastic T-cell lymphomas (AITLs). Recently, MAF contribution has been associated for the first time with breast cancer bone metastasis. In this thesis, we determined the role of MAF in several contexts. As a first approach we demonstrated that MAF is also a predictive marker of bone metastasis in prostate cancer (PC) patients. However, regarding androgen-independent PC cell lines, an overexpression of MAF was not enough to drive colonization of the mouse bone. Secondly, we report the beneficial effect of MAF downregulation on preventing skeletal metastasis in BoM2, a highly bone metastatic MCF7-derived cell line. MAF impinged bone colonization in a higher degree that other treatments against bone metastasis, such as PTHrP antagonist or recombinant OPG. This fact identifies MAF as a new potential target to focus on the generation of new drugs. Finally, MAF showed a tendency to redirect metastases to other organs than bone in the presence of ZOL treatment in vivo. Thus, we validated the association between MAF overexpression and an increase on extraskeletal metastases after ZOL preventive treatment in non-postmenopausal BC patients. Moreover, a mouse model was generated to better understand the biology of MAF- derived bone metastasis within complete stromal interactions. We designed a transgenic mouse model to express MAF in the mammary gland in an inducible manner. To this end, we generated two constructs; the first contains rtTA, renilla and katushka under MMTV promoter, and the second contains MAF, luciferase and tGFP under Tet-On promoter. We demonstrated the incorporation of several copy numbers of both transgenes in two independent colonies and we detected transgene expression under doxycycline activation by means of luminescent signal. Even though both colonies incorporated several copy number of the transgene, their expression was soft and some relevant leakiness was observed in the non-treated MAF mice. No differences were observed in terms of mammary gland development between transgene-expressing MAF mice and wild-type mice, as well as no tumor initiation was detected in any group. Notably, MAF Tg mouse was crossed with MMTV-PyMT to generate double Tg mice (PyMT-MAF). PyMT-MAF tumor growth presented no significant differences compared to PyMT in terms of time to tumor formation and growth rate. Importantly, no bone metastases were observed at 3-month-old mice of any group. Thus, the generation of this animal model provided new insights to generate a novel bone metastatic mouse model.
La identificació de gens implicats en el procés de la metàstasis és bàsica per tal d’entendre el mecanisme d’aquest procés, per reconèixer els pacients amb més risc de patir-lo i tractar-los selectivament i finalment pel desenvolupament de nous fàrmacs. Recentment, s’ha identificat el gen MAF com a predictor d’un alt risc de patir metàstasis òssia en pacients de càncer de mama. En aquesta tesi hem determinat el paper de MAF en diferents contexts. Per una banda, hem demostrat que MAF és un marcador predictiu de les metàstasis òssies també en pacients amb càncer de pròstata. Tot i així, una sobreexpressió de MAF en cèl·lules de càncer de pròstata andrògen-independents no va ser suficient per conduir la colonització a l’òs. Per altra banda, hem demostrat que reduir els nivells de MAF en les cèl·lules BoM2, derivades de les cèl·lules de càncer de mama MCF7, redueix la tendència d’aquestes cèl·lules a metastatitzar a l’òs. Cal destacar que aquest efecte és superior al d’altres tractaments com poden ser OPG recombinant o el pèptid antagonista de PTHrP, indicant MAF com a element potencial per a la generació de nous fàrmacs. Finalment, MAF afecta el patró de metàstasis de les cèl·lules ER- de càncer de mama després del tractament preventiu amb àcid Zoledronic, tal com s’observa en pacients. Per abordar el paper de MAF en el càncer de mama tenint en compte les interaccions amb l’estroma i el sistema immunitari, es va dissenyar un model animal transgènic que sobreexpressava MAF en la glàndula mamària de forma induïble. Es va demostrar la incorporació de vàries còpies del transgen en el ADN genòmic i també es va detectar, per senyal bioluminiscent, la inducció per doxiciclina de l’expressió del transgen. Cal destacar que l’expressió era feble i en molts casos inespecífica i independent al tractament amb doxiciclina. El desenvolupament mamari en aquest model no demostrava cap alteració com tampoc es va detectar cap indici de formació de tumor. Aquest model es va creuar amb MMTV-PyMT, i els tumors de les femelles doble transgèniques (PyMT-MAF) no van presentar cap diferència en el temps necessari per a la formació de tumors ni en la velocitat de creixement comparat amb PyMT. De manera destacable, no es van observar metàstasis òssies en el moment del sacrifici. D’aquesta manera, la generació del ratolí MAF transgènic ens va donar noves perspectives per enfocar la generació d’un nou model animal que generi metàstasis òssies.

Colorectal cancer is a common disease, representing the third and second most common cause of cancer death in the United States in women and men, respectively. [Ahnen et al.: Mayo Clin Proc 2014;89:216-224; Siegel et al.: CA Cancer J Clin 2016;66:7]. It is estimated that 20% of patients have distant metastatic disease at time of diagnosis [Ahnen et al.: Mayo Clin Proc 2014;89:216-224; Siegel et al.: CA Cancer J Clin 2016;66:7]. The most common metastatic sites include regional lymph nodes, liver, lungs, and peritoneum via lymphatic/hematogenous dissemination as well as contiguous and transperitoneal routes [Ahnen et al.: Mayo Clin Proc 2014;89:216-224; Siegel et al.: CA Cancer J Clin 2016;66:7]. Upon review of the literature, we found that metastatic colon cancer to the scrotum is rare. The following case report proved to be a unique example of this type of metastasis. This rare regional metastasis is theorized to have resulted from a colo-urethro-scrotal fistula that precipitated from the patient's prior traumatic event. (C) 2017 The Author(s) Published by S. Karger AG, Basel

Benign ovarian tumors and majority of epithelial ovarian cancers possess steroid receptors including estrogen receptors (ERs). However, the estrogen-ER signaling in ovarian carcinomas is not completely understood. Tumorigenesis is a multiple-step process involving dysregulated cell growth and metastasis. Tumor cells acquire the capacity of migration and invasion by temporal phenotypical and genotypical changes termed epithelial-mesenchymal transition (EMT). Considerable evidence implicates a mitogenic action of estrogen in early ovarian carcinogenesis. In contrast, its influence in the metastatic cascade of ovarian tumor cells remains obscure. In this study, I have focused on the role of 17β-estradiol (E2) in ovarian tumorigenesis. EMT related genes including E-cadherin, Snail, Slug, and Twist were examined. E2 treatment led to clear morphological changes and an enhanced cell migratory propensity. These morphologic and functional alterations were associated with changes in the abundance of EMT-related genes. Upon E2 stimulation, expression and promoter activity of the epithelial marker E-cadherin was strikingly suppressed, whereas EMT-associated transcription factors Snail and Slug were significantly up-regulated. This up-regulation was attributed to the increase in gene transcription activated by E2. Depletion of the endogenous Snail or Slug using small interfering RNA (siRNA) attenuated E2-mediated control in E-cadherin. In addition, the E2-induced cell migration was neutralized by Snail and Slug siRNAs, implying that both transcription factors are indispensable for the pro-metastatic actions of E2. Importantly, by using selective ER agonists as well as over-expression and siRNA approaches, it was identified that E2 triggered the metastatic behaviors exclusively through an ER⍺-dependent pathway. In contrast, overexpression of ERβ opposed the phenotypic changes and down-regulation of E-cadherin induced by ER⍺. In addition, microarray analysis was performed to characterize more putative downstream mediators of E2. Expression levels of 486 genes were found to be altered by at least 50% upon E2 treatment, and included several genes involved in oncogenesis, cell cycle control, apoptosis, signal transduction and the gene expression machinery. These candidate genes may be valuable for better delineating the ER pathways and functions. In summary, this study provides compelling arguments that estrogen can potentiate tumor progression by EMT induction, and highlight the crucial role of ER⍺ in ovarian tumorigenesis.

Malignant melanoma is the most lethal form of skin cancer due to the metastatic spread of the disease. Chemokines have been shown to play a key role in the metastatic cascade. The chemokine receptor CCR7 and its ligand CCL21 were shown to be involved in migration of CCR7 expressing melanoma cells towards a gradient of CCL21 released by lymphatic endothelial cells (LEC) and promote metastasis at the draining lymph node (LN) in in vivo tumour models. I therefore tested the hypothesis that growth of CCR7 -expressing tumours towards LEC is CCL21-dependent in vivo, that LECs promoted this growth through a chemokine-mediated mechanism without increasing the lymphatic clearance at the front of the tumour and that a recombinant human antibody, namely Chemotrap-1, that binds soluble CCL21, could inhibit chemokine-mediated lymphatic metastasis in vivo. I used in vitro migration assays in a modified Boyden chamber and an in vivo mouse model of directional tumour growth and tumour metastasis to determine the chemokine-mediated ability of melanoma cells to migrate and to test the effect of inhibitory antibodies. During the study on Chemotrap-1, I tested the binding affinity for the chemokine ligand CXCL 12, using specifically designed ELlSAs and tested the affinity for CXCL 12 in vitro and in vivo by migration assays. CCR7 was endogenously expressed in all melanoma cells examined, its overexpression promoted in transit metastasis in vivo and a CCL21 neutralising antibody inhibited its migratory potential. LEC depots in vivo did not promote any increase in lymphatic clearance that would have altered the directional tumour growth of subcutaneous melanoma tumours. Chemotrap-1 inhibited in vitro cell migration, LN metastasis in vivo and it also bound to CXCL 12. These results show the key role of the CCR7/CCL21 axis in lymphatic metastasis in malignant melanoma and highlight the therapeutic potential of Chemotrap-1 to target lymphatic metastasis CCL21- and CXCL 12-mediated.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2018.
DVD-ROM contains: movies/videos.
Cataloged from PDF version of thesis.
Includes bibliographical references.
Metastasis is the cause of the overwhelming majority of cancer deaths. However, it remains a poorly understood process. The events at the metastatic site are especially poorly comprehended. These events are dynamic and so require intravital imaging to investigate. However, the intravital imaging of these events in mice is challenging. Sites of metastasis are often in vital organs that are inaccessible to microscopy without surgical intervention. Furthermore, circulating tumor cells are rare and are involved in many transient interactions adding to the challenge. The development of a line of zebrafish, Casper, that is transparent throughout its life suggested that zebrafish might be a powerful system for intravital imaging. I first developed novel injection and imaging techniques to study metastasis through intravital imaging in adult zebrafish. I then followed individual ZMEL1 zebrafish melanoma cells at the metastatic site over the course of two weeks as they grew from single disseminated tumor cells into macroscopic metastases. From these studies, I characterized the steps of metastasis at the metastatic site for this cell line. I also utilized transparent zebrafish embryos to uncover a new role for the oncogene YAP during metastasis. I observed that the over-expression of a Hippo-insensitive mutant of YAP (YAP-AA) promoted brain metastasis following intravenous in zebrafish embryos. I determined that YAP-AA was promoting tumor cell dispersal throughout the embryo by allowing tumor cells to escape the first capillary bed they encounter. Following intravenous injection, control cells lodge in blood vessels in the tail and cease their travel through circulation. However, YAP-AA cells are able to move through these vessels, re-enter circulation and travel to other organs, such as the brain. These observations represent a new mechanism by which tumor cells can increase their dissemination throughout an animal.
by David Colin Benjamin.
Ph. D.

The majority of cancer related deaths occur due to the invasive growth of metastatic lesions. In the early stages of metastasis, circulating cell interact with the endothelial cells to establish at a distant site. In inflammation endothelial activation results in induction of adhesion molecules on the endothelium that participate in the homing of leukocytes. Because of the interactions of metastatic cells with the endothelium, the question was whether some of the characteristic molecules of endothelial activation were induced during metastasis. In vivo pulmonary metastatic models were used to characterize the expression profile of endothelial activation. Immunohistochemistry identified VCAM-1 to be induced on the pulmonary endothelium following tumour cell arrest. VCAM-1 upregulation was not observed prior to tumour cells arrest or within the first hours. In contrast, tumour cell arrest appeared to be required for endothelial activation, arguing against a mechanism analogous to leukocyte homing. The upregulation of VCAM-1 upon tumour cell arrest corresponded with the initiation of platelet clot formation around the tumour cell and recruitment of leukocytes to the site, both previously shown to be essential for metastasis. Disruption of both phenomena, either through genetic or pharmacological manipulation, demonstrated that in contrast to the recruited leukocytes, platelets were involved in inducing endothelial activation. Another protein investigated was VAP-1. In contrast to VCAM-1, central to VAP-1 adhesive function is its enzymatic activity. Blocking the functions of either molecule highlighted their role in facilitating the recruitment of the leukocyte population to the tumour cell. Disruption of which led to a significant attenuation of metastasis. While VCAM-1 and VAP-1 function appears critical in the early steps of metastasis, their inhibition had no effect at later stages of pulmonary colonization.

Da hochgradige Gliome nur eine geringe Tendenz zur Metastasierung aufweisen, beschränkte sich das klinische Wissen über diesen seltenen Krankheitsverlauf bisher im Wesentlichen auf die Erkenntnisse aus Einzelfallberichten und kleineren Fallserien. Eine detaillierte Analyse der beschriebenen Fälle war bisher nicht verfügbar. Die vorliegende Arbeit stellt eine systematische Auswertung der wissenschaftlichen Literatur über Patienten mit metastasierten Glioblastomen oder Gliosarkomen dar. Unser Ziel war es, sämtliche Publikationen zu berücksichtigen, welche bis April 2013 veröffentlicht worden sind. Mit Hilfe einer systematischen Literaturrecherche in den beiden Datenbanken PubMed und Web of Science konnten 215 Arbeiten identifiziert werden, welche insgesamt 357 Fallberichte enthielten. Die Prognose nach Diagnose einer Metastasierung ist infaust. In der untersuchten Patientenkohorte betrug die mediane Überlebenszeit lediglich 3.0 ± 0.4 Monate. Eine univariate Datenanalyse ergab, dass Geschlecht, Alter, der histologische Subtyp und das Zeitintervall zwischen der Diagnose des Primärtumors und der Metastasen die Überlebenszeit nicht beeinflussten. Im Gegensatz dazu war eine Metastasierung, die ausschließlich außerhalb des zentralen Nervensystems (ZNS) auftrat, mit längeren Überlebenszeiten verbunden. In den letzten Jahrzehnten wurden offenbar keine entscheidenden therapeutischen Fortschritte erzielt. Fälle, die in Publikationen bis zum Jahr 2000 Erwähnung fanden, wiesen keine schlechteren Überlebenszeiten auf als die nach der Jahrtausendwende publizierten Fälle. Aktuell gibt es keinen Datensatz, der geeignet wäre, die vielfältigen Therapieansätze systematisch auf ihre Wirksamkeit hin zu überprüfen. Wir sehen hier die Notwendigkeit, ein zentrales Register zu etablieren.

Cancer metastasis is the principal cause of death in patients with solid organ tumours. Once cancer has spread, it is generally considered incurable. This spread of cells from the primary tumour to distant sites (metastasis) is a complex process and this DPhil focuses on colorectal cancer (CRC) metastasis. It has three aims; the development of a new CRC metastasis model, the completion of a whole-genome screen for drivers of metastasis and the development of a greater understanding of the molecular drivers of metastasis through use of human lymph node metastasis (LNM) samples. The new CRC metastasis model is a mouse model utilising endoscope-guided orthotopic transplantation of tumour cells in combination with in-vivo imaging. This model has allowed the analysis of the role of the differentiation factor, Serum/Glucocorticoid regulated Kinase 1 (SGK1) in preventing metastasis and tumorigenesis. I provide preliminary data suggesting that the inhibition of metastasis by re-expression of SGK1 is associated with the down-regulation of the transcription factor Avian Myelocytomatosis Viral Oncogene Homolog (c-MYC), and low SGK1 is a poor prognosis biomarker identifying CRC patients with high risk of disease recurrence/death. A genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/cas9 screen was then performed to identify other genes that play important roles in CRC metastasis. The screen involved the orthotopic transplantation of the MC38 line in a mouse model of metastasis and subsequent analysis using targeted next generation sequencing. This approach identified the pioneer transcription factor, Forkhead Box F1 (FOXF1) as an important regulator of metastasis. I demonstrate that FOXF1 is down-regulated in human metastatic lesions, including LNM, liver and lung metastases. Metastasis driven by low FOXF1 is mediated through Mammalian Target of Rapamycin Complex (MTORC) signalling through transcriptional control of Regulatory Associated Protein of MTOR Complex 1 (RAPTOR). Finally, I performed an analysis of 69 stage III paired CRC tumour-LNM human specimens. Metastasis in this cohort was mediated through a number of important genes and gene networks, including C-X-C chemokine receptor type 4 (CXCR4) and Kirsten Rat Sarcoma Viral Oncogene (KRAS) signalling. Non-hierarchical clustering was performed and identified two distinct subsets of LNM, LNMS1 and LNMS2 each conferring different prognoses. I have also identified different immune profiles within the "Lymphoid ecological niche" using gene expression profiling. This has enabled me to demonstrate an association between a patient's immune profile, particularly activation of CD8 cells and KRAS signalling within the tumour, with improved disease free survival in stage III CRC patients.

Since lymph node stromal cells remain largely uncharacterized with respect to cell surface markers and function, their role in regulating the growth and invasion of disseminated cancer cells, including breast carcinoma has, to date, been virtually unexplored. In the present study, we asked whether peripheral lymph node cells could modulate the growth of breast carcinoma cells and, thereby, contribute to the progression of the metastatic process. Primary cultures of rat peripheral lymph node stromal cells were obtained by limiting dilution and two sublines, STA4 and STB12, with breast carcinoma growth-promoting activities were isolated. Immunocytochemistry performed on these cells revealed that they express vimentin, S-100 and fibronectin, but neither cytokeratin nor von Willebrand factor indicating that they are stromal and dendritic in origin. Several functional studies were performed using media conditioned by STA4 and STB12 cells. (Abstract shortened by UMI.)

Breast cancer is the most frequently diagnosed and the second leading cause of cancer deaths in Canadian women. The most devastating and deadly feature of the disease is the emergence of metastases. Breast cancer most commonly metastasizes to bone, often leading to a significantly decreased quality of life in affected patients. Despite progress in understanding the underlying molecular biology of breast tumors that relapse to bone, to date there are no therapies capable of curing the disease. Hence, it is essential to gain a more in-depth knowledge of the molecular mechanisms that underlie the emergence and growth of breast cancer skeletal metastases. Consequently, it was attempted to: 1) examine the efficacy of targeting a known pathway important for breast cancer metastasis to bone, 2) identify novel mediators of this process and 3) develop a stratification tool capable of identifying patients with breast cancer that possesses a high likelihood of spreading to bone. Transforming growth factor-beta (TGF-β) signaling is a potent modulator of the invasive and metastatic behavior of breast cancer cells. The work in this thesis demonstrates that expression of a TGF-β ligand trap, which neutralizes TGF-β1 and TGF-β3 in breast cancer cells, diminished their outgrowth in bone and reduced the severity of osteolytic lesion formation. It is further shown that a reduction or loss of host-derived TGF-β1 reduced the incidence of breast tumor outgrowth in the skeleton. Moreover, tumor cells capable of growing within the bone of a TGF-β1 deficient host up-regulated expression of all three TGF-β isoforms within the tumor cells themselves, effectively bypassing the host-deficiency. Next, a gene discovery approach was undertaken to identify novel candidate mediators of breast cancer skeletal metastasis. Invasive breast epithelium was selectively isolated by laser capture microdissection (LCM) performed on bone metastases and primary tumors from patients displaying breast cancer with subsequent recurrence to the skeleton. In this search, ABCC5 was found to be overexpressed in osseous metastases compared to primary mammary tumors metastatic to bone. Furthermore, this protein was detected at substantially higher levels in human and mouse breast cancer cells, which metastasize to bone in animal models. Importantly, removal of this protein from these cells resulted in their decreased ability to induce osteolytic bone lesions, which was correlated with a decreased recruitment of osteoclasts, cells responsible for the bone resorption process. Finally, the molecular changes occurring within the primary breast tumor were investigated in an attempt to identify a prognostic bone metastatic signature. Gene expression profiling was performed on estrogen receptor (ER)-positive primary breast tumors metastastatic to bone and breast cancers, which spread to soft tissue. A 25-gene signature was derived from the top 100 differentially expressed probes and was found to be capable of discriminating breast tumors metastatic to bone from cancers recurring to visceral sites in an independent gene expression dataset.
Le cancer du sein est le cancer le plus fréquemment diagnostiqué et la deuxième cause de décès par cancer chez les femmes canadiennes. La caractéristique la plus dramatique et la plus mortelle de cette maladie est l'apparition de métastases. Le cancer du sein métastase le plus souvent au niveau des os, conduisant fréquemment à une qualité de la vie sensiblement diminuée chez les patients atteints. Malgré les progrès réalisés dans la compréhension de la biologie moléculaire sous-jacente des tumeurs du sein qui colonisent l'os, il n'existe, à ce jour, aucun traitement capable de guérir cette affection. Il est ainsi primordial d'acquérir une connaissance plus approfondie des mécanismes moléculaires qui sont à l'origine de l'émergence et de la croissance des métastases du cancer du sein au niveau du squelette. Par conséquent, il fut envisagé: d'examiner l'efficacité du ciblage d'une voie connue, importante pour que le cancer du sein puisse métastaser au niveau de l'os; d'identifier les nouveaux médiateurs de ce processus et de développer un outil de discrimination capable d'identifier les patients atteints d'un cancer du sein qui possède une forte probabilité de dissémination à l'os. La voie de signalisation du facteur de croissance transformant bêta (TGF-β) est un puissant modulateur du comportement invasif et métastatique des cellules cancéreuses du sein. Le travail présenté dans cette thèse démontre que l'expression d'une molécule leurre du ligand du TGF-β, qui neutralise le TGF-β1 et le TGF-β3 dans les cellules cancéreuses du sein, réduit leur développement dans les os et ainsi que la gravité de la formation des lésions ostéolytiques. Il est également démontré qu'une réduction, ou une perte, de l'hôte dérivé du TGF-β1 réduit la fréquence d'apparition d'une excroissance de la tumeur mammaire au niveau du squelette. Par ailleurs, les cellules tumorales capables de croître au sein d'un tissu osseux hôte déficient en TGF-β1 ont montré une augmentation de l'expression des trois isoformes du TGF-β dans les cellules tumorales elles-mêmes, court-circuitant ainsi efficacement cette carence de l'hôte. Une approche de découverte de gènes a été ensuite entreprise pour identifier des nouveaux médiateurs candidats des métastases squelettiques des cancers du sein. L'épithélium invasif du sein a été sélectivement isolé par microdissection à capture laser (LCM), et ceci a été réalisé sur les métastases osseuses et des tumeurs primaires de patientes présentant un cancer du sein avec récidive ultérieure au niveau du squelette. Dans cette recherche, ABCC5 fut montré comme étant surexprimé dans les métastases osseuses, par rapport à des tumeurs primaires mammaires métastasant au niveau l'os. De plus, cette protéine a été détectée à des niveaux nettement plus élevés dans des cellules cancéreuses mammaires d'origine humaine et murine qui métastasent dans l'os dans des modèles animaux. Une autre donnée importante fut que la suppression de cette protéine dans les cellules concernées conduisit à une réduction de leur capacité à induire des lésions osseuses ostéolytiques, ce qui fut corrélée avec une diminution du recrutement d'ostéoclastes, les cellules responsables du processus de résorption osseuse. Pour terminer, les changements moléculaires qui se produisent au sein de la tumeur primaire du sein ont été étudiées dans le but d'identifier une signature pronostiquant des métastases osseuses. Un profilage de l'expression génique a été réalisé sur les tumeurs mammaires primaires positives pour le récepteur aux œstrogènes (ER) et métastasant à l'os mais aussi sur les cancers mammaires, qui se propagent aux tissus mous. Une signature de 25 gènes a été sélectionnée à partir des 100 meilleures sondes exprimées différentiellement et a montré sa capacité à discriminer d'un coté les tumeurs du sein métastasant à l'os et de l'autre les cancers récurrents sur des sites viscéraux et ceci à partir d'un ensemble de données sur l'expression de gènes indépendants.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2016.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references.
Adhesion-GPCRs, a novel family of G protein-coupled receptors (GPCRs), are characterized by an extended extracellular region linked to a seven-pass transmembrane moiety via GPCR proteolytic site (GPS)-containing stalk region known as GAIN domain. The name adhesion refers to the presence of functional domains in the extracellular region that commonly mediate cell-cell and cell-matrix interactions in various contexts. Recently, many genome-scale analyses of genetic alterations across diverse cancer types have revealed significant alterations (copy number and mutational) in adhesion-GPCRs, yet no comprehensive examination of their roles in cancer biology exists. Through a systematic screening for all adhesion-GPCRs by RT-qPCR in murine mammary carcinoma cell lines with varying metastatic abilities as well as tumor samples of different grades, I have identified several candidate genes with possible roles in breast cancer progression and metastasis. Based on these analyses and cross-referencing with the published gene expression data on human breast cancer cell lines and patient samples, I chose two candidate genes, CELSR2 and GPR126, for more detailed investigation. To elucidate their functions in cancer biology, I investigated the effects of their perturbations using RNAi (loss-of-function) methods both in vitro and in vivo. The results from my work reveal that loss of CELSR2 affects neither tumor growth nor lung metastasis in a xenograft mouse model of breast cancer, despite enhancing invadopodial activity in vitro. I also show that highly metastatic breast cancer and melanoma cells have elevated levels of GPR126, and confirm the significance of this result by revealing (a) reduction in pulmonary metastasis without affecting primary tumor growth in a spontaneous metastasis model of breast cancer, and (b) reduction in lung metastasis in three different experimental metastasis models of breast cancer and melanoma, upon shRNA-mediated knockdown of GPR126. After probing the different steps in the metastatic cascade to investigate how GPR126 promotes metastasis, I demonstrate that GPR126 specifically affects extravasation, most likely through its engagement with type IV collagen in the sub-endothelial basement membrane. Thus, the work described in this thesis contributes to our overall understanding of the perplexing problem of cancer metastasis via identification of novel regulators of distinct steps along the ominous path of malignant cells from primary sites to distant organs.
by Bigyan R. Bista.
Ph. D.

The spread of cancer to distant sites, metastasis, is responsible for the majority of cancer related deaths. Metastasis is a complex process comprising a number of steps and mechanisms which take place over time. Due to the temporal and extemperaneous nature of metastasis it has proved difficult to study. Current models are limited in their application and there is a need to develop new models which provide a better and more meaningful biological context for the study of metastasis. Taking a tissue engineering approach, this project has sought to develop a set of in vitro models for the exploration of cancer metastasis in three dimensions. Two collagen based assays were developed to allow the exploration of metastasis in a three dimensional (3D) environment. A simple collagen based assay was developed to create multiple regions of interest, allowing the study of cell migration, invasion and colonization in two dimensions, three dimensions and at border zones. Compression of collagen was used to construct a stiffer more elastic 3D in vitro context and this assay was developed to provide multiple regions of interest for the study of metastasis in a more structured and biologically relevant environment. The chick chorioallantoic membrane (CAM) assay was explored as an in vivo model for the study of metastasis however this allowed only a short time period for study. However, the decellularization of CAM tissue provided a novel and useful 3D context which could be used for the study of metastatic mechanisms in direct comparison to the in vivo model but over longer time periods.

Progression to metastatic disease is the leading cause of deaths resulting from breast cancer. Understanding the mechanisms underlying a cell's ability to move away from its site of origin and populate a distant site is important for the future development of therapies. The interactions between a tumor cell and the microenvironment can modulate a cell's ability to invade through tissues and access distant organs. In this study we present evidence indicating the differential modulation of invasive and proliferative phenotypes by hyaluronan present in the cellular microenvironment.We establish the role of CD44, the primary receptor for hyaluronan, in breast cancer progression and metastasis through the use of transgenic mouse models of breast cancer. While no differences were seen in the onset of primary breast tumors, mice expressing CD44 had a reduced rate of pulmonary metastasis compared to mice that lacked CD44. This establishes an anti-invasive role for CD44 in breast tumor progression. We also identify a decreased population of alveolar macrophages in CD44 negative mice that could affect metastatic breast cancer cell colonization of the lungs.We then focused our study in vitro, where we assessed the invasive properties of breast cancer cells as they move through three dimensional (3D) matrices containing or lacking hyaluronan. We show that in 3D type I collagen gels, breast cancer cells invade more readily in the absence of hyaluronan compared to when hyaluronan (HA) is embedded within the gel. HA mediated inhibition of invasion is dependent on CD44 binding as demonstrated through the use of a CD44 functional blocking antibody.We also show that HA promotes differential phenotypes of breast cancer cell. HA promotes filopodia formation and invasion when soluble in the cell microenvironment. Alternatively, matrix-embedded HA inhibits invasion and promotes migration through the formation of lamellipodia. The differential HA invasive and proliferative phenotypes are mediated by differential activation of ERK or γPAK. Activation of γPAK is mediated by CD44 while ERK activation by HA occurs by CD44 independent mechanisms.We also demonstrate an inhibition of MMP9 mediated invasion by HA when embedded within a type IV collagen matrix, but not a type I collagen matrix. This differential activity indicates that it is not only the immobilization of HA in a matrix that determines its activity, but also the context in which it is present within the matrix.These data underscore the importance of studying matrix components in an environment that closely resembles in vivo conditions. HA is a prime example as it has the capability of both promoting and inhibiting invasion depending on how it is presented to a cell. Differential HA activity also underlies the importance of understanding extracelluar matrix degradation and the release of matrix components as these can adversely affect disease progression.

The dissemination of tumor cells to distant organs i.e. metastasis is an exceedingly complex process leading to 90% of all cancer deaths. Despite being so clinically important, little is known about this process that requires tumor cells to leave the primary tumor site, intravasate and transport through the blood stream, extravasate and colonize at secondary sites leading to distant metastases. Survivin, a member of the IAP (Inhibitor of Apoptosis) family with known functions in apoptosis and mitosis, is highly expressed in aggressive tumors and is associated with poor prognosis and adverse clinical outcome. But the mechanistic role of survivin in metastatic dissemination has not been investigated. In this study, we demonstrate an important and novel role of survivin in activating a broad gene expression program in tumor cells. Of particular importance is the upregulation of a distinct class of cell adhesion molecules, particularly fibronectin. This IAP mediated gene regulation requires synergistic intermolecular cooperation between survivin and its related cofactor molecule, XIAP that results in activation of NF-κB dependent fibronectin gene expression. The binding of fibronectin with its cognate cell surface receptors initiates outside–in signaling leading to the autocrine and paracrine activation of cell motility kinases, FAK and Src, in turn leading to enhanced tumor invasion and metastasis. The importance of survivin and XIAP in the process of metastasis has also been demonstrated in vivousing intrasplenic injections in mouse models. Overall this study is the first to place survivin upstream of transcriptional activation of gene expression particularly fibronectin. In addition, it also demonstrates the importance of survivin-XIAP complex in mediating NF-κB activation which in turn switches on the expression of various target genes involved in tumor metastasis. Hence this study dissects the upstream and downstream requirements of survivin- XIAP complex mediated tumor dissemination and metastasis. Significance of this Study The hallmark of end-stage cancer is metastasis, an incurable condition almost invariably associated with death from disease. Despite a better understanding of the metastatic process, and the identification of key gene expression requirements of this pathway, the development of anti-metastatic therapies has lagged behind, with no viable options being currently offered in the clinical setting. Our findings that Inhibitor of Apoptosis (IAP) proteins functions as metastasis-promoting genes independently of cell survival, but through activation of cell motility could have important ramifications for the broader application of IAP antagonists currently in early clinical trials, as novel anti-metastatic therapies.

Breast cancer is the most common non-skin cancer in women and the second most common cause of cancer-related death in U.S. women. Despite numerous advances in treatment strategies against breast cancer, the presence of undetected distant metastasis of the primary tumor remains the main cause of mortality. Current screening and detection methods such as mammograms are simply not sensitive enough to detect formation of metastasis. Further, currently available therapies against metastatic breast cancer do not provide a complete cure for the disease. Thus, understanding the biology and molecular factors involved in cancer metastasis will help aid in preventing the onset of metastasis and discovering an effective treatment for this deadly disease. My research focused on understanding the mechanism of action of one such factor, breast cancer metastasis suppressor 1 (BRMS1), a suppressor gene found deleted in late stage breast cancers. The goal of my dissertation was to investigate the role of membrane signaling lipids phosphoinositides, specifically phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) in BRMS1-mediated metastasis suppression in MDA-MB-435 and MDA-MB-231 human breast carcinoma cells. My studies revealed BRMS1 selectively reduced receptor tyrosine kinases (RTK) and Gprotein coupled receptors (GPCR) expression and downstream signaling in human breast carcinoma cells. My observations are critical as many of these receptors are upregulated in metastatic breast cancer and PI(4,5)P2 is a critical constituent for mediating their downstream signaling events. Further, using immunoblotting studies, I uncovered a possible compensatory mechanism in tumor cells to overcome downregulation of PI(4,5)P2 by BRMS1 and maintain its downstream signaling. When studied for BRMS1 regulation of enzymes involved in PI(4,5)P2 synthesis, I showed BRMS1 completely inhibits phosphatidylinositol 4-phosphate 5-kinase β (PIP5Kβ) expression. Using overexpression studies, I showed PIP5Kβ to be the major contributor to the cellular PI(4,5)P2 pool required for agonist-induced intracellular calcium rise. Taken together, my dissertation research has identified some critical breast cancer markers and revealed signaling pathways altered by BRMS1 in human breast carcinoma cells that can be studied as potential therapeutic targets against breast cancer metastasis.

Accompanying CD-ROM contains image files and software. Bibliography: leaves 259-286. Explores the factors affecting the movement of tumour cells from a primary malignancy across the peritoneal cavity to the port-site following laparoscopic intervention. Filter methods and radio-labelled tumour cells provided the most useful way of following cell movement. Concludes spread of tumour cells to the port-site is more likely in the presence of disseminated disease, as well as with inappropriate surgical technique. Metastasis may be reduced by the use of intraperitoneal lavage and appropriate surgical technique.

MACC1, ein Hauptregulator von Metastasen, ist an zahlreichen Kennzeichen von Krebs beteiligt, einschließlich dereguliertem Metabolismus. Dennoch ist seine Rolle im Krebsstoffwechsel unklar. In der vorliegenden Arbeit wurde eine systematische Analyse von MACC1-getriebenen metabolischen Netzwerken durchgeführt. MACC1 erhöhte die GLUT1 auf Zellmembrane, was zu einer erhöhten Glukoseanreicherung, einem erhöhten Glukosefluss und somit zu einer erhöhten Zellproliferation führte. Außerdem, reduzierte MACC1 den Glutaminfluss unabhängig von der Nährstoffverfügbarkeit. Bei Glucoseentzug erhöhte MACC1 die Pyruvataufnahme und zeigte aber die geringe Auswirkungen auf den Pyruvatfluss. In vivo, MACC1 zeigte erhöhte Aufnahme von 18F-FDG und 18F-Glutamat in Lebermetastasen. Zusammengefasst zeigen diese Ergebnisse, dass MACC1 mehrere Wirkungen auf den Krebs-Metabolismus zeigt, was es attraktiv macht, seine Wirkungen in Krebsmodellen weiter zu untersuchen. Metastasierung ist die Haupttodesursache bei Darmkrebs. Fünfzehn bis zwanzig Prozent der Darmkrebs Patienten im Stadium II entwickeln im Verlauf der Erkrankung Metastasen, jedoch bleiben die Kriterien der Wahrscheinlichkeit mit welcher Patienten von Chemotherapie profitieren werden ungenau. Hier wurde das Potenzial der Plasma-Metabolomik zur Vorhersage von Metachronmetastasen untersucht. Plasma metabolische Profile wurden wesentlich unterschiedlich zwischen nicht-metastasierten und metachron metastasierten Patienten gefunden. Wie Klassifikationsmodelle aus Entscheidungsbäumen und Support-Vektor-Maschinen gezeigt haben die Plasmametaboliten haben die Fähigkeit nicht-metastasierte von metachron metastasierten Darmkrebs Patienten zu unterscheiden, mit einer durschnittlichen Vorhersagegenauigkeit von 0,75 bzw. 0,82 für jede der Methoden, angemessen. Zusammen, zeigen diese Ergebnisse, dass Plasmametaboliten das Potenzial haben, Darmkrebs Patienten gemäß ihrem Metastasierungsrisiko nichtinvasiv zu stratifizieren.
MACC1, a master regulator of metastasis, is involved in most hallmarks of cancer, including deregulated metabolism. Yet, fragmentary data on its role in cancer metabolism exist. Here, a systematic analysis of MACC1-driven metabolic networks by elucidation of cell nutrient preferences, environment dependent alterations of nutrient utilization, metabolic pathway functionality and metabolic tracing using 13C-labeled metabolic substrates had been performed. MACC1 was found to enhance surface GLUT1 thus leading to increased glucose depletion, glucose flux and hence increased cell proliferation. Besides, MACC1 was found to reduce glutamine flux independent of nutrient availability. Upon glucose deprivation MACC1 was found to enhance pyruvate uptake exhibiting minor effects on pyruvate flux. In vivo, MACC1 increased uptakes of 18F-FDG and 18F-glutamate in liver metastatic lesions. Together, these findings demonstrate that MACC1 exhibits multiple effects on cancer metabolism, thus making it attractive to further study its effects in cancer models. Metastasis is the main cause of death from colorectal cancer (CRC). Fifteen to twenty percent of stage II CRC patients develop metastasis during the course of disease, however the criteria of likely benefitting patients from chemotherapy remain imprecise. Here, the potential of plasma metabolomics to predict metachronous metastasis was assessed. Plasma metabolic profiles were shown to be significantly different between non-metastasized and metachronously metastasized CRC patients. As demonstrated by supervised classifications using decision trees and support vector machines plasma metabolites have the power to distinguish non-metastasized from metachronously metastasized CRC patients giving average prediction accuracy of 0.75 and 0.82 for each of the methods, respectively. Together, these results demonstrate that plasma metabolites have the potential to non-invasively stratify CRC patients according to their metastasis risk.

We studied the mechanisms driving the metastatic spread in colorectal cancer (CRC), focusing in the MAPK pathway. We developed in vivo a highly metastatic cell line using a KRAS-mutated cell line (SW620) in an ortothopic xenograft mouse model and used both in vivo and in vitro experiments to evaluate the mechanisms of metastasis. We also used data from two large prospective cohorts of incident CRC to evaluate the association of an intronic variant of SMAD7 (rs4939827, 18q21) with the phenotype and molecular characterisitics of CRC. In the first project, we inoculated SW620 luciferase-expressing cells into portal circulation of immunodeficient mice via intrasplenic injection followed by splenectomy, in order to isolate cell populations that target the liver. Comparative transcriptomic analysis identified 194 genes differentially expressed between the parental and the highly metastatic cell line. We found pathways of nitrogen metabolism, cell adhesion molecules and mitogen-activated protein kinases (MAPKs). Downregulation of ERK2 (but not of ERK1) in the highly metastatic cell line reverted its metastatic capacity to the liver, but not to the lung in our mice model. We thus hypothesized that the ability to metastasize the lung by the highly metastatic derivative had to be driven by other mechanism. The expression of parathyroid hormone-like hormone (PTHLH) was upregulated in our highly metastatic derivative and was inversely correlated with the expression of MKK6. Downregulation of PHTLH in the highly metastatic derivative decreased its capacity to colonize the lung without decreasing its capacity to colonize the liver after intra portal inoculation. We evidenced that PTHLH induced apoptosis of human pulmonary endothelial cells via apoptosis-inducing factor mitochondrion-associated 1. In the second project we evaluated the association of the SMAD7 intronic variant with tumor phenotype and several CRC molecular characteristics. We used 1509 CRC cases and 2307 age-matched controls nested within the Nurses Health Study (NHS) and the Health Professionals Follow-up Study (HPFS). We randomly selected between one and three matched controls. Among the 1509 cases with blood or buccal samples in this study, we were able to successfully obtain tissue suitable for molecular analyses in 658 cases. We genotyped rs4939827 (TaqMan) successfully in 98% of the samples in NHS and 99.6% of the samples in HPFS. The phenotipic features evaluated were: TNM stage, grade of differentiation, location of the primary tumor (colon vs. rectum) and age at diagnosis. The evaluated molecular characteristics were DNA methylation of RUNX3 and LINE-1 (long interspersed nucleotide element-1), CpG island methylator phenotype (CIMP), microsatellite instability, TP53 expression by immunohistochemistry and the mutational status of BRAF, KRAS and PIK3CA. We found that the minor allele (G) in rs4939827 was associated with a lower risk of developing tumor stage pT1 or pT2 CRC [multivariate odds ratio (OR), 0.73; 95% confidence interval (CI) 0.62-0.87] but not tumor stage pT3 or pT4 (multivariate OR, 1.07; 95% CI 0.93-1.23, P for heterogeneity = 1.2 x 10-4). The association between rs4939827 and CRC also significantly differed by methylation of RUNX3 (P for heterogeneity = 0.005). Among those with CRC, the minor allele (G) in rs4939827 was significantly associated with poorer overall survival (hazards ratio, 1.20; 95% CI, 1.02-1.42). In conclusion, we provide clinical and molecular evidence showing that ERK2 activation provides colon cancer cells with the ability to seed and colonize the liver and reduced p38 MAPK signalling endows cancer cells with the ability to form lung metastasis from previously established liver lesions. We also show that patients with the rs4939827 CRC-susceptibility locus diagnosed with CRC tend to develop tumors with greater invasiveness (as measured by the pT stage).
Parte de esta investigación consiste en explorar los mecanismos involucrados en el patrón metastático de CCR. Asimismo usamos datos epidemiológicos donde evaluamos la asociación entre el polimorfismo intrónico de SMAD7 (rs4939827, 18q21) con el genotipo y características tumorales. En el primer proyecto usamos un modelo murino de metástasis hepáticas para crear un derivado celular con alto tropismo metastático a hígado y pulmón. Mediante análisis de expresión de genes usando chips de transcripción identificamos 194 genes diferencialmente expresados El análisis de muestras clínicas mostró que aquellos pacientes cuyo tumor presentaba bajos niveles de p38 sufrían una mayor frecuencia de metástasis al pulmón, pero no a otros órganos. Al tratar ratones que habían desarrollado metástasis hepáticas derivadas de la línea celular parental con un inhibidor específico de p38, vimos que se incrementaba la afinidad metastática al pulmón. Evidenciamos que p38, a través del silenciamiento de PTHLH, en el derivado celular altamente metastático disminuía su capacidad de colonizar el pulmón. Demostramos que PTHLH induce la apoptosis de células humanas de endotelio pulmonar a través del factor AIFM1, facilitando que las células metastáticas puedan extravasarse al pulmón. En el segundo proyecto evaluamos la asociación de un polimorfismo intrónico del gen SMAD7 con el fenotipo y varias características moleculares del tumor. Para ello empleamos 1509 casos de cáncer de colon y recto y 2307 controles emparejados anidados en las cohortes Nurses Health Study y Health Professionals Follow-up Study. Encontramos que el alelo de menor frecuencia de rs4939827 (G) se asociaba con un menor riesgo de desarrollar un CCR con un estadio pT1 o pT2 [razón de odds (OR) ajustada, 0.73; intervalo de confianza al 95\% (CI) 0.62-0.87] pero no con tumores con estadio pT3 o pT4 (OR ajustada, 1.07; 95\% CI 0.93-1.23, valor p de heterogeneidad = 1.2 x 10-4). La asociación entre el polimorfismo de rs4939827 y CCR también difería significativamente según la metilación de RUNX3 (valor p de heterogeneidad = 0.005). Entre aquellos pacientes diagnosticados con CCR, el alelo de menor frecuencia de rs4939827 (G) estaba significativamente asociado con peor supervivencia (hazards ratio, 1.20; 95\% CI, 1.02-1.42).

Thesis (Ph. D.)--University of Missouri-Columbia, 2005.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "December 2005" Includes bibliographical references.

[Truncated abstract] Melanoma cell adhesion molecule (MCAM) is highly expressed in more than 70% of metastatic melanoma and is correlated with invasive potential. However, the specific contribution MCAM makes to invasion and metastasis in melanoma is not clear. In this study, I have demonstrated that transfection of MCAM into MCAM-negative melanoma and CHO cells leads to changes in cell shape, and the modulation of cell-to-cell and cell-matrix interactions. MCAM positive cells were slower to spread on collagen type I, collagen type IV and laminin 1 than MCAM negative cells, although these differences were not apparent on vitronectin, fibronectin and laminin 10. In contrast, MCAM expression had little effect on cell adhesion to any of the matrices tested. MCAM positive (compared to negative) cells also showed morphological changes and a rearrangement of the actin cytoskeleton when plated on a matrix containing laminin 5. Taken together, these data suggest that MCAM expression modulates β1-integrinmediated spreading on matrix, but has little effect on αvβ3-mediated cell-matrix interactions. As this study provided little evidence to suggest that MCAM transfection altered β1 integrin expression levels on melanoma cells, it is proposed that a competitive interaction between the cytoplasmic domains of MCAM and β1 integrin may affect mature focal adhesion assembly. MCAM expression in melanoma cells was also associated with decreased cell movement over matrix into a scratch-wound site and an increased tendency to form cell cords on Matrigel. These two assays gauge the propensity of a cell to engage in cell-cell versus cell-matrix interactions, and suggest that MCAM positive cells favour cell-cell adhesion. Interestingly, MCAM transfection was also associated with an increased ability of melanoma cells to migrate through a basement membrane towards a chemoattractant. ... Analysis of the intracellular domain of MCAM revealed the presence of tyrosine and dileucine endocytosis signals. Interestingly, disruption of these two motifs did not seem to impair the internalization of MCAM from the cell surface. The di-leucine motif, however, was necessary for the recycling of MCAM back to the surface following endocytosis. Lastly, MCAM was found to exists as dimers within the cell membrane in the absence of ligand, although the exact location of the dimerization motif is not yet clearly defined. Collectively, findings from my study suggest: MCAM expression in melanoma cells facilitates cell-cell interactions, whilst concomitantly modulating cell-matrix interactions. MCAM transfection also leads to enhanced migration of melanoma cells through a basement membrane. Thus, MCAM expression may increase the ability of melanoma cells to migrate as a collective, a feature of highly invasive cancer. The intracellular domain of MCAM interacts with ApxL2, a novel member of the Shroom family of actin-binding proteins. It is likely that ApxL2 links a proportion of MCAM within the cell to the actin cytoskeleton, contributing to cell shape determination and other processes, such as migration. MCAM exists as dimers on the cell surface and is internalized at least partially by a clathrin-mediated mechanism.

Metastasis is thought to be based on genetic and epigenetic alterations. The mechanisms underlying prostate cancer metastasis are not clear. Studies aimed at identifying genes with key roles in this process have been impeded by lack of clinically relevant models. The heterogeneity of primary prostate cancer specimens from patients, consisting of non-metastatic and metastatic subpopulations, hampers identification of metastasis-associated genes by direct comparison of primary and secondary cancers. To overcome such hurdles, metastatic and non-metastatic tumor sublines have been developed from one patient’s primary prostate cancer specimen using subrenal capsule grafting into NOD-SCID mice. Chromosomal alterations present in the metastatic subline, but not in non-metastatic counterparts, were identified in a small percentage of cells in the parental tissue, suggesting that metastatic potential of primary cancers can be associated with a small cancer cell subpopulation. Sublines with different metastatic potential derived from same patient’s multifocal primary cancer provide valuable materials for identifying metastasis-associated genes and predictive markers. To identify metastasis-associated genes, differential gene expression analysis of metastatic PCa1-met and non-metastatic PCa2 prostate cancer sublines was carried out. Among various differentially expressed genes identified, ASAP1, a gene not previously associated with prostate cancer, was upregulated in the metastatic subline as confirmed by qRT-PCR and immunohistochemical staining. In clinical specimens, ASAP1 protein staining was elevated in 80% of primary prostate cancers and substantially higher in metastatic lesions compared to benign prostate tissue. Extra ASAP1 gene copies were detected in 58% of primary prostate cancer specimens. Increased ASAP1 protein expression was correlated with prostate cancer metastasis and PSA recurrence. siRNA- and shRNA-induced reduction of levels of ASAP1 protein markedly suppressed in vitro PC-3 cell migration, matrigel invasion and metastasis in vivo. These results indicate that ASAP1 plays an important role in prostate cancer invasion and metastasis and suggest that it provides a potential predictive marker and therapeutic target for the disease. Furthermore, the approach used to identify metastasis-associated genes by comparison of gene profiles of paired metastatic and non-metastatic sublines was validated. The subrenal capsule xenograft system provides a valuable platform for studying various aspects of prostate cancer metastasis.

Introduction Squamous cell carcinoma of the auricle has an unusually high rate of lymph node metastases when compared to similar tumours at other sites. The lymph nodes affected are close to the base of the skull and in the neck. Development of metastasis carries a poor prognosis and most patients will subsequently die of failure of loco-regional control. Despite the likelihood of a poor outcome nothing can be done for patients prior to development of metastasis, as the risk of spread is not sufficiently high to warrant intervention in all patients. They are therefore treated with a ‘wait and see policy’ and only offered treatment once clinical evidence of metastatic spread is detected. This thesis sets out to examine what can be done, at the time of initial presentation with an auricular squamous cell carcinoma to identify patients who would benefit from treatment to the regional lymph node basins. Materials and Methods The thesis is divided into four separate studies. A systematic review examines the evidence available to date, an anatomical study examines the lymphatic drainage of the auricle in cadavers, a sentinel lymph node biopsy study examines the use of this technique to identify early tumour spread and a retrospective analysis of cases of auricular squamous cell carcinoma in our unit examines histopathological prognostic indictors of metastatic spread. Results The systematic review found that these tumours have a metastatic rate of about 11%. Patients developing metastasis usually die from failure of loco-regional control. Depth of tumour invasion, tumour size and mode of invasion seem to be potential indicators of metastatic risk. There is a strong argument for prophylactic intervention to the regional lymph nodes but there is no consensus of opinion as to when this should be carried out The anatomical study comprised 5 cadaveric dissections. They showed that the first echelon nodes draining the auricle lie in the superficial parotid gland, post-auricular/ mastoid nodal group and level II of the neck. There are anastamotic pathways around the mastoid and post-auricular nodes that could permit embolic tumour cells to bypass them. Five lymphatic pathways draining the auricle are described and some of these lie on the lateral and anterior surfaces of the mastoid bone and traverse the insertion of sternocleidomastoid. 28 cases of auricular squamous cell carcinoma were enrolled for sentinel lymph node biopsy. None of them were found to have any metastatic spread. One case showed non-viable tumour cells in a lymph node. There was a high incidence of complications (14%) directly related to the sentinel node biopsy procedure. The retrospective analysis identified 229 cases of auricular squamous cell carcinoma treated in our unit from 1992 - 2004. 212 of these cases had the primary pathology available for analysis. 24 (of 212) patients developed metastasis. 17 patients died as a result of their disease usually due to failure of control at the regional lymph node basin. Primary tumours with a depth of invasion greater than 8mm have metastatic rate of 56%. Tumours with a depth of invasion between 2-8mm and evidence of cartilage destruction, lymphovascular invasion or a non-cohesive invasive front have 24% metastatic rate. Tumours outwith these high-risk groups did not metastasise. Conclusions Elective lymph node dissections of the superficial parotid gland, post-auricular/mastoid and level II nodes should be considered in patients with primary auricular squamous cell carcinomas with a depth of invasion >8mm or a depth of invasion between 2 - 8 mm and evidence of cartilage destruction, lymphatic invasion or a non-cohesive invasive front. This should ideally be done as part of an observational study to evaluate the cost / benefit ratio for these patients. The neck dissection must clear the mastoid bone to a sub-periosteal level on its anterior and lateral surfaces. This will require the removal of the upper portion of sternocleidomastoid. Sentinel lymph node biopsy requires further study to evaluate it as a method for early detection of metastatic spread in auricular squamous cell carcinoma. This could be done as part of an observational study of elective neck dissections.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2004.
Includes bibliographical references (leaves 108-124).
Metastasis, or the spread of a primary tumor to distal sites in the body, is the major cause of human cancer-related morbidity and mortality. Metastasis requires a complex series of cellular events that remain poorly understood at the molecular level. Recently, advances in microarray technology have allowed cancer biologists to globally survey metastatic progression and define patterns of gene expression that correlate with progression to a metastatic phenotype. By using such a genomic approach, our laboratory identified a subset of genes that regulate the actin cytoskeleton whose enhanced expression correlates with metastasis. This thesis describes the characterization of IQGAP1, a key regulator of the cytoskeleton, as a potentially critical player in metastatic progression. Here I show a strong positive correlation between IQGAP1 expression levels and metastatic progression in both in vivo-selected human metastatic melanoma cells and other human tumors. In addition, I have experimentally analyzed the role of IQGAP1 in metastasis using two different dominant-negative mutants. The results suggest that IQGAP1 may play a functional role in metastatic progression, particularly in the processes of cell migration and invasion. This work lays a scientific framework by which cancer biologists can look at global gene expression analyses and then probe deeper into individual genes to define the molecular mechanisms underlying their roles. In addition, this work contributes to a deeper understanding of the molecular pathogenesis of metastasis, and identifies in IQGAP 1 a potential molecular target for future tumor metastasis therapies.
by Sarah Elizabeth Ann Frew.
Ph.D.

Tese de Doutoramento em Ciências Veterinárias, especialidade de Ciências biológicas e biomédicas
The metastatic spread of cancer is still the major barrier to the treatment of this disease. Cancer-mortality is mainly due to recurrence and metastasis. Although much has been done in the field of cancer treatment, prevention and approach to metastases are still areas not fully explored. Over and under-expression of Dll4/Notch signaling has been demonstrated to impair tumor growth through opposing patterns of vascular modulation in different mouse tumor models and human cancer xenografts. Recent evidence implicates Dll4/Notch pathway in the metastasis mechanism, but less is known about the specific role of endothelial Dll4. For this reason, we proposed to investigate how endothelial Dll4 expression interferes with the metastatic process. To address it we used a spontaneous metastasis mouse model based on Lewis Lung Carcinoma (LLC) subcutaneous transplants in endothelial-specific Dll4 lossof- function (eDll4cKO) and endothelial-specific Dll4 over-expression (D4OE) mice. Results demonstrated that eDll4cKO is responsible for the vascular function regression that leads to tumor growth reduction. Early steps of epithelial-to-mesenchymal transition (EMT) and cancer stem cell selection were also inhibited by eDll4cKO, leading to a substantial reduction of circulating tumor cells and reduction in the number and burden of macrometastases. Intravasation and extravasation were also compromised by eDll4cKO, possibly due to blockade of the metastatic niche. In the case of the D4OE mice we observed that tumor growth reduction was achieved by vessel proliferation restriction along with an improved vascular maturation, which allowed a more efficient delivery of chemotherapy. This last effect of vessel normalization seemed to prevent metastasis formation even though EMT markers were increased. In conclusion, despite the opposite vascular architecture phenotypes of eDll4cKO and D4OE, both lead to a reduction in metastasis. This is in line with the concept of Dll4 dosage observed in the wound-healing context and represents a promising therapeutic approach in metastasis prevention/ treatment.
RESUMO - A importância da expressão endotelial do ligando Dll4 no processo de metastização - O cancro é, atualmente, uma das principais causas de morte em todo o mundo, a par das doenças cardiovasculares e da doença pulmonar obstrutiva crónica. A mortalidade por cancro está frequentemente associada à metastização, quer seja pelo compromisso hemodinâmico, quer seja pelas consequências do seu tratamento. Apesar dos avanços no tratamento do cancro, com novos agentes biológicos e imunomodeladores, a investigação do mecanismo da metastização, prevenção e abordagem das metástases continuam a ser áreas pouco exploradas. A via Notch é uma via de sinalização intercelular altamente conservada que está envolvida na determinação do destino, regulação da proliferação e diferenciação celulares. O ligando Dll4 tem uma expressão predominantemente endotelial arterial e capilar, crítica para o correto desenvolvimento embrionário, mas restringindo-se às pequenas artérias e capilares no adulto em homeostasia. Ele atua como agente anti-angiogénico em situações de “stress” fisiológico e em contexto tumoral, promovendo a ocorrência de pausas ritmadas na fase de crescimento vascular ativo, que são conducentes à maturação funcional da vasculatura nascente. Os efeitos do aumento e diminuição da expressão do ligando Dll4 sobre a dinâmica tumoral já foram extensamente estudados em modelos tumorais de ratinhos transgénicos e com xenografos. Geralmente, a ativação aberrante da via de sinalização Dll4/Notch está associada a mau prognóstico e probabilidade de metastização. Por outro lado, as terapias de bloqueio Dll4/Notch têm-se revelado eficazes no tratamento de modelos tumorais resistentes às terapias baseadas no “vascular endotelial growth factor” (Vegf), em modelos pré-clínicos. Estudos recentes implicam a ativação da via Notch nas células endoteliais, com alteração do seu fenótipo de forma a favorecer a transmigração das células tumorais, bem como a secreção de “vascular cell adhesion molecule-1”, que promove a adesão de leucócitos e potencia os fenómenos de intravasação e extravasação. Nesse sentido, surge a oportunidade de explorar o papel do ligando Dll4, em concreto, de que forma é que a sua expressão endotelial influencia as várias etapas da cascata da metastização. Para isso, usamos um modelo murino de metastização espontânea que consistiu na injeção subcutânea de células Lewis Lung Carcinoma (LLC) em dois tipos de ratinhos transgénicos: ratinhos com perda-de-função ou sub-expressão endotelialespecífica de Dll4 e ratinhos com ganho-de-função ou sobre-expressão endotelialespecífica de Dll4. Relativamente ao estudo da influência do ganho-de-função endotelialespecífico de Dll4 sobre o tumor primário, foram também usados outros ratinhos: o modelo de papiloma da pele induzido quimicamente e o modelo murino transgénico RIP-Tag2, com insulinoma. Para avaliar a angiogénese do tumor primário foram determinados parâmetros como: densidade, maturidade, funcionalidade e permeabilidade vasculares. Depois de explorado o efeito de Dll4 sobre o fenómeno de transição-epitélio-mesenquimal (EMT), in vitro, procurou-se explorar o mesmo, in vivo, com recurso aos ratinhos mutantes endoteliais-específicos citados. EMT foi caracterizada através da marcação imunofluorescente para Snail-1 e Twist. As células tumorais estaminais (CSC) foram exploradas através da marcação imunofluorescente para p63 e CD49f. A hipóxia tumoral foi avaliada através do teste com “Hypoxyprobe”. O circuito das células tumorais metastáticas foi analisado com recurso à marcação das células com uma proteína verde fluorescente, recorrendo às técnicas de imunofluorescência e “fluorescent associated cell sorting”. A análise da expressão génica foi feita através do “quantitative real time polymerase chain reaction”, tanto nos ratinhos com perda, como ganho-de-função endotelial-específico de Dll4. Quanto ao estudo da perda-de-função endotelial-específica de Dll4 foi possível avaliar, genericamente, todas as etapas da metastização. Começando pelo tumor primário, verifica-se que ocorre uma redução considerável do seu crescimento, em parte devido à angiogénese que, apesar de aumentada, é desorganizada e menos funcional. A árvore vascular apresenta inúmeras ramificações e um revestimento peri-vascular insuficiente (redução de células de músculo liso, dos marcadores α-SMA e Pdgfr-β), indicativo de imaturidade vascular. Observa-se um aumento da permeabilidade, que leva a fenómenos de exsudação e consequente má perfusão do tumor. Este endotélio disfuncional acaba por sofrer regressão, condicionando o crescimento tumoral. Constata-se que eventos iniciais como a EMT e a seleção de clones de células tumorais estaminais (CSC) são inibidos pelo bloqueio endotelial-específico de Dll4, conduzindo a uma redução do número de células tumorais circulantes e do número e crescimento das macro-metástases. Os fenómenos posteriores de intravasação e extravasação estão também reduzidos, provavelmente pela não sinalização do tumor primário à medula óssea, via Dll4/ Notch1/ Hey1/ TGF-β. Pelo que, há uma redução da mobilização de células mieloides Cd11c+/VEGFR-1+ para o nicho metastático (pulmão) e fraca deposição de fibronectina, que seria essencial para a acomodação das células tumorais circulantes/metastáticas. De salientar, que não ocorre sinalização parácrina entre o endotélio pulmonar e as células tumorais metastáticas, já que não se verifica marcação para N1ICD pulmonar. No caso da avaliação do papel do ganho-de-função endotelial-específico de Dll4, verificouse que a redução do crescimento tumoral resulta duma diminuição da angiogénese (com redução concordante da expressão dos receptores pro-angiogénicos Vgfr1 e Vgfr2), mas também da sua normalização. Trata-se duma árvore vascular com poucas ramificações, com características de maturidade evidenciadas pelo grande revestimento peri-vascular (aumento de células de músculo liso, dos marcadores α-SMA, Pdgfr-βe Ephrin-B2), permitindo um fluxo sanguíneo eficiente, ainda que inferior ao normal, pela redução da densidade vascular. Este efeito, aparentemente paradoxal (contra-intuitivo), acaba por promover um melhor aporte da doxorrubicina (quimioterápico), com bloqueio do crescimento e/ou indução da apoptose tumoral. Esta normalização parece também inibir a metastização, já que apesar de haver um aumento da expressão dos marcadores de EMT, essas células tumorais acabam por ficar “aprisionadas” no endotélio tumoral, dado que se constata uma redução do número de macro-metástases. No entanto, este modelo carece de clarificação no número de células tumorais circulantes, bem como nas restantes etapas da metastização que não chegaram a ser exploradas. Em ambos os modelos, verifica-se que é a função endotelial Dll4/Notch, e não a hipóxia tumoral, que determina a sinalização para EMT e seleção de CSC. Concluindo, observa-se no contexto da dinâmica tumoral fenótipos opostos para os modelos de perda e ganho-de-função endotelial-específico de Dll4, em linha com o que já foi descrito para o contexto da cicatrização de feridas. Estamos novamente perante o conceito de dose-dependência de Dll4, com aparente repercussão também na metastização. Isto porque o fenótipo final é o mesmo, de redução do número de macrometástases, apesar das diferenças na angiogénese tumoral e no evento inicial de EMT, ficando por explorar a restante cascata da metastização. O uso do ligando Dll4, na forma de sub ou sobre-expressão, pode constituir uma nova abordagem terapêutica ao tratamento dos cancros da mama e colo-rectal metastizados, a par de outros anti-angiogénicos já utilizados. Mas mais interessante será explorar a sua abordagem como fármacos antagonistas ou agonistas, numa estratégia de prevenção da metastização.
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