Malgré le rôle des variants constitutivement actifs du récepteur des androgènes (RA) et du stroma tumoral dans le cancer de la prostate résistant à la castration (CRPC), leurs relations restent inconnues. Nous rapportons l’impact de l’interleukine-6 (IL-6) sécrétée par les cellules stromales prostatiques (PrSC) sur les cellules épithéliales tumorales prostatiques exprimant les variants autonomes du RA. Le milieu de culture conditionné par les PrSC (CMPrSC) contenait des taux élevés d’IL-6 et induisait une augmentation de l’activité transcriptionnelle de STAT3 dans les LNCaP et C4-2b exprimant le variant RAQ640X, via une activation de pY705-STAT3. Cette activité de STAT3 était inhibée par la neutralisation de l’IL-6. L’analyse par mRNA array et RT-qPCR a mis en évidence un profil transcriptomique spécifique lié à l’expression du RAQ640X et à l’exposition au CMPrSC, impliquant les fonctions de motilité, d’invasion et de migration cellulaires, et l’expression de gènes favorisant la dissémination métastatique. Ainsi, nos résultats illustrent une coopération épithélio-stromale «privilégiée» en présence de variants autonomes du RA, impliquée dans la progression tumorale. / Constitutively active androgen receptor (AR) variants and stromal microenvironment are involved in castration resistant prostate cancer (CRPC), but their relationship remains unknown. We describe the effects of interleukin-6 (IL6) secreted from prostate stromal fibroblast cells (PrSC) towards prostate epithelial cancer cells expressing constitutively active AR variants. Conditioned culture medium from PrSC (CMPrSC) contained high levels of IL-6 and led to an increased STAT3 transcriptional activity in LNCaP and C4-2b cells expressing the ARQ640X variant, through pY705-STAT3 activation. This STAT3 activity was significantly diminished with neutralizing antibody anti-IL6. Gene expression analysis using mRNA array and RT-qPCR highlighted a specific transcriptional profile related to ARQ640X expression and PrSC exposure, resulting in cellular motility, invasion and cellular migration, and IL-6 genes expression promoting metastatic dissemination. Overall, our data emphasize a “preferred” epithelio-stromal cooperation when expressing constitutive active RA variants, which contributes to tumor progression.

Lung cancer leads in mortality among all types of cancer in the US and Non-small cell lung cancer (NSCLC) is the major type of lung cancer. Immuno-compromised mice bearing xenografts of human lung cancer cells represent the most common animal models for studying lung cancer biology and for evaluating potential anticancer agents. However, orthotopic lung cancer models based on intrapulmonary injection of suspended cancer cells feature premature leakage of the cancer cells to both sides of the lung within five days, which generates a quick artifact of metastasis and thus belies the development and progression of lung cancer as seen in the clinic. Based on intrapulmonary inoculation of multicellular spheroids (MCS), we have developed the first orthotopic xenograft model of lung cancer that simulates all four clinical stages of NSCLC progression in mice over one month: Stage 1 localized tumor at the inoculation site; Stage 2 multiple tumor nodules or larger tumor nodule on the same side of the lung; Stage 3 cancer growth on heart surface; and Stage 4 metastatic cancer on both sides of the lung. The cancer development was monitored conveniently by in vivo fluorescent imaging and validated by open-chest anatomy, ex vivo fluorescent imaging, and histological studies. The model enjoys high rates of postoperative survival (100%) and parenchymal tumor establishment (88.9%). The roughness of the inoculated MCS is associated negatively with the time needed to develop metastatic cancer (p=0.0299). In addition, we have constructed a co-culture MCS that consisted of A549-iRFP lung cancer cells and WI38 normal human fibroblast cells. The pro-proliferation effect and the high expression of α-smooth muscle actin (α-SMA) by the co-cultured WI38 cells indicated their transformation from normal fibroblasts to cancer-associated fibroblasts (CAFs). The morphology of the co-culture MCS features a round shape, a tight internal structure, and quicker development of roughness. The large roughness value of co-culture MCS suggests that small co-culture MCS could be inoculated into mice lung with a small needle to reduce the surgical trauma. Taken together, a new orthotopic model of NSCLC has been developed, which would facilitate future development of medications against lung cancer.

Cancer metastasis, the spread of cancer from the primary site to distant regions in the body, is the major cause of cancer mortality, accounting for almost 90% of cancer related deaths. During metastasis, cancer cells from the primary tumor initially probe the surrounding fibrous tumor microenvironment (TME) prior to detaching and subsequently migrating towards the blood vessels for further dissemination. It has widely been acknowledged that the biophysical cues provided by the fibrous TME greatly facilitate the metastatic cascade. Consequently, there has been a tremendous wealth of work devoted towards elucidating different modes of cancer cell migration. However, our knowledge of how cancer cells at the primary tumor site initially sense their fibrous surroundings prior to making the decision to detach and migrate remains in infancy. In part, this is due to the lack of a fibrous in vitro platform that allows for precise, repeatable manipulation of fiber characteristics. In this study, we use the non-electrospinning, Spinneret based Tunable Engineered Parameters (STEP) technique to manufacture suspended nanofiber networks with exquisite control on fiber dimensions and network architecture and use these networks to investigate how single cancer cells biophysically sense fibers mimicking in vivo dimensions. Using high spatiotemporal resolution imaging (63x magnification/1-second imaging interval), we report for the first time, that cancer cells sense individual fibers by coiling (i.e. wrapping around the fiber axis) at the tip of a cell protrusion. We find that coiling dynamics are mediated by both the fiber curvature and the metastatic capacity of the cancer cells with less aggressive cancer cells showing diminished coiling. Based on these results, we explore the possibility of using coiling in conjunction with other key biophysical metrics such as cell migration dynamics and forces exerted in the development of a genetic marker independent, biophysical predictive tool for disease progression. Finally, we identify the membrane curvature sensing Insulin Receptor tyrosine kinase Substrate protein of 53 kDa (IRSp53) as a key regulator of protrusive activity with IRSp53 knockout (KO) cells exhibiting significantly slower protrusion dynamics and diminished coil width compared to their wild-type (WT) counterparts. We demonstrate that the hindered protrusive activity ultimately translates to impaired contractility, alteration in the nucleus shape and slower migration dynamics, thus highlighting the unique role of IRSp53 as a signal transducer – linking the protrusive activity at the cell membrane to changes in cytoskeletal contractility. Overall, these findings offer novel perspectives to our understanding of how cancer cells biophysically sense their fibrous surroundings. The results from this study could ultimately pave the way for elucidating the precise fiber configurations that either facilitate or hinder cancer cell invasion, allowing for the development of new therapeutics in the long term that could inhibit the metastatic cascade at a relatively nascent stage and yield a more promising prognosis in the perennial fight against cancer. / Doctor of Philosophy / Cancer is a leading cause of death worldwide. Almost ninety percent of cancer related deaths arise from the spreading of cancer cells from the primary tumor site to secondary sites in the body – a processed termed as metastasis. The environment surrounding a tumor (tumor microenvironment) is highly fibrous in nature and can assist in the metastatic process by providing biophysical cues to the cells at the tumor boundary. These cells sense the presence of the surrounding fibers by extending "arms" termed as protrusions, and then eventually detach from the primary tumor and start migrating through the fibrous microenvironment. While numerous studies have investigated the various modes of cell migration in fibrous environments, there is very little information regarding how cancer cells use protrusions to initially sense the fibers prior to detaching. In this study, we used the Spinneret based Tunable Engineered Parameters (STEP) technique to manufacture suspended nanofiber networks with robust control on fiber diameter and network architecture and use these networks to systematically investigate how single cancer cells biophysically sense fibers that mimic in vivo dimensions. We discovered that cancer cells sense individual fibers by "wrapping-around" the axis of the fiber at the tip of the protrusion – a phenomenon we refer to as coiling. We found both the fiber diameter as well as the invasive capacity of cells can influence the coiling mechanics. Based on these results, we explored the use of coiling in conjunction with other key biophysical metrics such as the cell migration speed and how much force a cell can exert to develop a biophysical predictor for cancer cell aggressiveness. Finally, given that cells sense the fiber curvature by coiling, we explored the role of a key curvature sensing protein Insulin Receptor tyrosine kinase Substrate protein of 53 kDa (IRSp53) in mediating coiling activity and found that knocking out (KO) IRSp53 results in reduced coiling and slower protrusions compared to wild-type (WT) cells. Furthermore, IRSp53 KO cells showed impaired contractility which led to an alteration in the nucleus shape and slower migration dynamics thus highlighting the role of IRSp53 in linking changes at the cell membrane to the underlying cell cytoskeleton. The results from this study could ultimately help us understand what type of fiber conditions around a primary tumor would either help or delay the emergence of the tumor boundary cells and thus allow for the development of therapeutics that could significantly slow down the metastatic process at a relatively early stage.

Le cancer épithélial de l’ovaire (CEO) est le cancer gynécologique le plus létal. Plus de 70% des patientes diagnostiquées avec une tumeur de stade avancé rechutent suite aux traitements chimiothérapeutiques de première ligne, la survie à cinq ans étant ainsi très faible. Afin de mieux comprendre l’évolution de la maladie, nous avons recherché de nouveaux gènes, responsables de l’initiation et de la progression du CEO. Précédemment, des lignées cellulaires ont été dérivées à partir de la tumeur primaire et récurrente et/ou d’ascites de trois patientes. Le séquençage de l’ARN de ces lignées par la technologie de séquençage de nouvelle génération (TSNG) nous a permis d’identifier des mutations ponctuelles qui pourraient nous indiquer des gènes dérégulés dans le CEO. La TSNG est un bon outil qui permet d’identifier et de cribler à grande échelle des mutations. Nous avons sélectionné PLEC1, SCRIB, NCOR2, SEMA6C, IKBKB, GLCE et ITGAE comme gènes candidats présentant des mutations dans nos lignées et ayant une relation fonctionnelle avérée avec le cancer. Étant donné que la TSNG est une technique à taux de fiabilité limité, nous avons validé ces mutations par séquençage Sanger. Ensuite, nous avons étudié l’effet de ces mutations sur la structure protéique et l’expression de PLEC1, de SCRIB et de SEMA6C. Seules certaines mutations dans les gènes PLEC1, SCRIB et SEMA6C ont pu être confirmées. PLEC1 et SCRIB sont deux protéines d’échafaudage dont la mutation, rapportée dans plusieurs cancers, pourrait induire des changements de leurs conformations et affecter leurs interactions et leurs fonctions. Les conséquences de ces mutations sur la tumorigenèse de l’ovaire devront être étudiées. / Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer. Over 70% of the patients diagnosed with advanced stage of cancer relapse following first-line chemotherapy treatments; consequently the five-year survival is very low. To better understand the evolution of the disease, our aim was to identify new genes responsible for the initiation and progression of EOC. Previously, cell lines derived from solid tumors or ascites were developed from the primary and recurrent tumor or ascites of three patients. RNA sequencing of these cell lines by next-generation sequencing technology (NGST) allowed us to identify mutations that might point to genes whose deregulation is important in EOC. Mutations were detected in PLEC1, SCRIB, NCOR2, SEMA6C, IKBKB, GLCE and ITGAE. We selected these genes for further studies as they have previously been identified as being associated with cancer. First, we validated these mutations by Sanger sequencing in order to determine the concordance with NGST data. Secondly, we studied the impact of the validated mutations on protein structure and gene expression. Only certain mutations in PLEC1, SCRIB and SEMA6C were confirmed. Of interest, PLEC1 and SCRIB are two scaffold proteins, where mutations have been reported in several cancers and, possibly leading to changes in their conformation and thereby affecting their interactions and functions. The consequences of these mutations on ovarian tumorigenesis remain to be determined.

Le cancer épithélial de l’ovaire (CEO) est le cancer gynécologique le plus létal. Plus de 70% des patientes diagnostiquées avec une tumeur de stade avancé rechutent suite aux traitements chimiothérapeutiques de première ligne, la survie à cinq ans étant ainsi très faible. Afin de mieux comprendre l’évolution de la maladie, nous avons recherché de nouveaux gènes, responsables de l’initiation et de la progression du CEO. Précédemment, des lignées cellulaires ont été dérivées à partir de la tumeur primaire et récurrente et/ou d’ascites de trois patientes. Le séquençage de l’ARN de ces lignées par la technologie de séquençage de nouvelle génération (TSNG) nous a permis d’identifier des mutations ponctuelles qui pourraient nous indiquer des gènes dérégulés dans le CEO. La TSNG est un bon outil qui permet d’identifier et de cribler à grande échelle des mutations. Nous avons sélectionné PLEC1, SCRIB, NCOR2, SEMA6C, IKBKB, GLCE et ITGAE comme gènes candidats présentant des mutations dans nos lignées et ayant une relation fonctionnelle avérée avec le cancer. Étant donné que la TSNG est une technique à taux de fiabilité limité, nous avons validé ces mutations par séquençage Sanger. Ensuite, nous avons étudié l’effet de ces mutations sur la structure protéique et l’expression de PLEC1, de SCRIB et de SEMA6C. Seules certaines mutations dans les gènes PLEC1, SCRIB et SEMA6C ont pu être confirmées. PLEC1 et SCRIB sont deux protéines d’échafaudage dont la mutation, rapportée dans plusieurs cancers, pourrait induire des changements de leurs conformations et affecter leurs interactions et leurs fonctions. Les conséquences de ces mutations sur la tumorigenèse de l’ovaire devront être étudiées. / Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer. Over 70% of the patients diagnosed with advanced stage of cancer relapse following first-line chemotherapy treatments; consequently the five-year survival is very low. To better understand the evolution of the disease, our aim was to identify new genes responsible for the initiation and progression of EOC. Previously, cell lines derived from solid tumors or ascites were developed from the primary and recurrent tumor or ascites of three patients. RNA sequencing of these cell lines by next-generation sequencing technology (NGST) allowed us to identify mutations that might point to genes whose deregulation is important in EOC. Mutations were detected in PLEC1, SCRIB, NCOR2, SEMA6C, IKBKB, GLCE and ITGAE. We selected these genes for further studies as they have previously been identified as being associated with cancer. First, we validated these mutations by Sanger sequencing in order to determine the concordance with NGST data. Secondly, we studied the impact of the validated mutations on protein structure and gene expression. Only certain mutations in PLEC1, SCRIB and SEMA6C were confirmed. Of interest, PLEC1 and SCRIB are two scaffold proteins, where mutations have been reported in several cancers and, possibly leading to changes in their conformation and thereby affecting their interactions and functions. The consequences of these mutations on ovarian tumorigenesis remain to be determined.

This diploma thesis includes an experimental and a bioinformatic part. The two parts are linked together through the subject of transcription factors of 'forkhead box O' (FOXO) family. FOXO transcription factors have a key role in many cellular processes including cell cycle regulation, apoptosis and metabolism. For a long time, they have been considered strictly as the tumor-suppressors yet a growing number of evidence is pointing out to their pro-tumorigenic role. In consequence FOXO transcription factors are studied intensively as potential therapeutic targets in cancer. In the past decade, in silico prediction of protein-protein interactions has become popular in basic research as well as in drug development. Nonetheless, the predicted structures are still far from fitting to the expected behavior of the respective biomolecules. In the experimental part of this thesis, I verified the interaction of four in silico predicted protein binders based on naturally occurring PDZ domain with FOXO4 using microscale thermophoresis. Non-invasive bladder tumors represent a heterogeneous disease where reliable prediction of tumor aggressiveness is still lacking despite an intensive research. In the bioinformatic part of this thesis, I described the cellular composition of the tumor microenvironment and demonstrated...

Indiana University-Purdue University Indianapolis (IUPUI) / Although rare, classification of over 200 hereditary cancer susceptibility syndromes accounting for ~5-10% of cancer incidence has enabled the discovery and understanding of cancer predisposition genes that are also frequently mutated in sporadic cancers. The need to prevent or delay invasive cancer can partly be addressed by characterization of cells derived from healthy individuals predisposed to cancer due to inherited "single-hits" in genes in order to develop patient-derived samples as preclinical models for mechanistic in vitro studies. Here, we present microarray-based transcriptome profiling of Li-Fraumeni syndrome (LFS) patient-derived unaffected breast epithelial cells and their phenotypic characterization as in vitro three-dimensional (3D) models to test pharmacological agents. In this study, the epithelial cells derived from the unaffected breast tissue of a LFS patient were cultured and progressed from non-neoplastic to a malignant stage by successive immortalization and transformation steps followed by growth in athymic mice. These cell lines exhibited distinct transcriptomic profiles and were readily distinguishable based upon their gene expression patterns, growth characteristics in monolayer and in vitro 3D cultures. Transcriptional changes in the epithelial-to-mesenchymal transition gene signature contributed to the unique phenotypes observed in 3D culture for each cell line of the progression series; the fully transformed LFS cells exhibited invasive processes in 3D culture with disorganized morphologies due to cell-cell miscommunication, as seen in breast cancer. Bioinformatics analysis of the deregulated genes and pathways showed inherent differences between these cell lines and targets for pharmacological agents. After treatment with small molecule APR-246 that restores normal function to mutant p53, we observed that the neoplastic LFS cells had reduced malignant invasive structure formation from 73% to 9%, as well as an observance of an increase in formation of well-organized structures in 3D culture (from 27% to 91%) by stereomicroscopy and confocal microscopy. Therefore, the use of well-characterized and physiologically relevant preclinical models in conjunction with transcriptomic profiling of high-risk patient derived samples as a renewable laboratory resource can potentially guide the development of safer and more effective chemopreventive approaches.

At present, prostate cancer is a significant public health issue throughout the world and is the second leading cause of cancer deaths in older men. The prostate specific antigen or PSA (which is encoded by the kallikrein 3/KLK3 gene) test is the current most valuable tool for the diagnosis and management of prostate cancer. However, it is insufficiently sensitive and specific for early diagnosis, for staging of prostate cancer or for discriminating between benign prostatic hyperplasia (BPH) and prostate cancer. Recent research has revealed another potential tumour marker, glandular kallikrein 2 (KLK2 gene/hK2 protein), which may be used alone or in conjunction with PSA to overcome some of the limitations of the PSA test. Twelve new kallikrein gene family members have been recently identified and, like hK2 and PSA, many of these genes have been suggested to be involved in carcinogenesis. In this study, the cellular localisation and level of expression of several of these newer kallikreins (KLK4, KLK5, KLK7, KLK8 and KLK11) was examined in prostate tissue, to provide an understanding of the association of their expression with prostatic diseases and their potential as additional biomarkers. Like PSA and hK2, the present observation indicated that each of these proteins, hK4, hK5, hK7, hK8 and hK11, was detected within the cytoplasm of the secretory cells of the prostate glands. For the first time, all of these newly-identified proteins were shown to be expressed in prostatic intraepithelial neoplasia (PIN) lesions, in comparison to normal glands and cancer lesions. In addition to cytoplasmic secretory cell expression, the localisation of hK4 to the basal cells and nuclei in prostatic lesions was intriguing. The intensity of hK4 staining in prostate tissue was strongest in comparison to the other newly-identified kallikrein proteins (hK5, hK7, hK8 and hK11). Therefore, KLK4/hK4 expression was characterised further to define this cellular localisation and examined in non-prostatic tissue and also in a larger number of prostate tissues in an attempt to determine its potential value as a biomarker for prostate disease. Three hK4 antipeptide polyclonal antibodies, derived against N-terminal, mid-region and C-terminal hK4 amino acid sequences, were used. The hK4 N-terminal antipeptide antibody was used to demonstrate the cellular localisation of hK4 in kidney, salivary glands, liver, testis, colon carcinoma, heart, endometrium and ovarian cancer, for the first time. The presence of hK4 in these non-prostate tissues was consistent with the previous reports using RT-PCR. The dual cytoplasmic and nuclear localisation of hK4 observed in the prostate above was also seen in these tissues. Although hK4 was found widely expressed in many human tissue types, indicating that it is not prostate specific in its expression, the highest expression level of hK4 was seen in the prostate. Therefore, detailed expression patterns and levels of KLK4 mRNA and hK4 protein in the normal prostate and prostatic diseases and histopathological lesions were investigated and reported for the first time in this study. Twelve benign prostatic hyperplasia (BPH), 19 adenocarcinoma (Gleason grade 2-5) and 34 bone metastases from prostate cancer were analysed. Using in situ hybridisation, the expression of KLK4 mRNA was detected in the cytoplasm of the secretory cells of both normal and diseased prostate tissue. KLK4 mRNA was also noted in both secretory and basal cells of PIN lesions, but the basal cells of normal glands were negative. Using the hK4 N-terminal and mid-region antipeptide antibodies, hK4 was predominantly localised in the cytoplasm of the secretory cells. The intensity of hK4 staining appeared lowest in normal and BPH, and increased in PIN lesions, high Gleason grade prostate cancer and bone metastases indicating the potential of hK4 as a histopathological marker for prostatic neoplasias. Further studies are required with a larger cohort to determine its utility as a clinical biomarker. Small foci of atypical cells, which were found within normal glands, were also intensely stained. Surprisingly, hK4 protein was found in the nucleus of the secretory cells (but not the basal cells) of high grade PIN and Gleason grade 3 prostate cancer. The detection of KLK4 mRNA and hK4 protein in PIN lesions and small foci of atypical cells suggests that up-regulation of KLK4 expression occurs early in the pathology of prostate carcinogenesis. The finding of basal cell expression is not typical for the kallikreins and it is not clear what role hK4 would play in this cell type. With the use of the hK4 C-terminal antipeptide antibody, the staining was mainly localised in the nuclei of the secretory cells of the prostate glands. Although the nuclear localisation was readily noted in more than 90% of epithelial cells of the prostate gland with the C-terminal antibody, no difference in staining intensity was observed among the histopathological lesions of the prostate. The prominent nuclear localisation with the C-terminal antipeptide antibody was also shown to be distributed throughout the nucleus by using confocal microscopy. Further, by using gold-labelled particles for electron microscopy, the intracellular localisation of these hK4 antipeptide antibodies was reported here for the first time. Similar to the immunohistochemical results, the cytoplasm was the major site of localisation with the N-terminal and mid-region antipeptide antibodies. To further characterise the involvement of KLK4/hK4 in human prostate cancer progression, the transgenic adenocarcinoma mouse prostate (TRAMP) model was used in this study. In this study, mouse KLK4 (also known as enamel matrix serine protease -1, EMSP-1) was shown to be expressed in the TRAMP prostate for the first time. Previous studies had only shown the developing tooth as a site of expression for EMSP-1. The level of EMSP-1 mRNA expression was increased in PIN and prostate cancer lesions of the TRAMP model, while negative or low levels of EMSP-1 mRNA were seen in normal glands or in control mouse prostate tissue. The normal mouse prostate did not stain with any the three hK4 antipeptide antibodies. hK4 N-terminal and mid-region antipeptide antibodies showed positive staining in the cytoplasm of the epithelial cells of PIN and cancer lesions of the mouse prostate. The C-terminal antipeptide antibody showed distinctively nuclear staining and was predominantly localised in the nuclei of the glandular cells of PIN and cancer lesions of the mouse prostate. The expression patterns of both the mRNA and protein level for mouse KLK4 strongly supported the observations of KLK4/hK4 expression in the human prostate and further support the utility of the TRAMP model. Overall, the findings in this thesis indicate a clear association of KLK4/hK4 expression with prostate cancer progression. In addition, several intriguing findings were made in terms of cellular localisation (basal as well as secretory cells; nuclear and cytoplasmic) and high expression in atypical glandular cells and PIN, perhaps indicating an early involvement in prostate disease progression and, additionally, utility as basal cell and PIN histological markers. These findings provide the basis for future studies to confirm the utility of hK4 as a biomarker for prostate cancer progression and identify functional roles in the different cellular compartments.