No description available.

Pancreatic Ductal Adenocarcinoma (PDAC) affects approximately 8000 people every year in the UK and is the fifth leading cause of cancer related death. At a molecular level PDAC is characterized by a significant immune infiltrate. Tumour-associated macrophages (TAMs) infiltrate the tumour and contribute to a worse prognosis by promoting growth, metastasis and resistance to chemotherapy. TAMs are derived from circulating ‘classical’ CD14++ CD16- monocytes in the peripheral blood. Current work in murine models suggests targeting monocyte recruitment in PDAC can reduce TAM infiltration and disease burden therefore improving survival. This project aims to identify markers specific to monocytes from PDAC patients and to investigate their biological relevance and potential for therapeutic intervention. Gene expression and metabolomics analysis was carried out on classical CD14++ CD16- monocytes from locally advanced PDAC patients and age matched healthy donors. Transcriptomic profiling revealed a significantly altered gene expression profile in classical monocytes from patients and genes with the highest fold change difference were chosen for validation using qPCR. Validated gene targets were investigated further in vitro and large-scale gene expression analysis from pancreatic tumours assessed. The results from my work demonstrate that the gene expression profile of classical monocytes from PDAC patients is significantly different compared to healthy volunteers. Identification and validation of up-regulated genes and their biological relevance may represent a relevant novel novel biomarker or therapeutic strategyies to target monocytes and myeloid recruitment in cancer.

High-throughput mass spectrometry has discovered a plethora of candidates in the biomarker field, however, subsequent verification and validation studies are urgently needed to assess the potential of novel biomarkers in the detection of pancreatic cancer. We have conducted extensive verification and validation studies on two of our most promising biomarkers CUZD1 and LAMC2 with a total of 715 blood samples. In our study, both markers demonstrated consistent diagnostic ability of early- and CA19.9 negative-PDAC cases. When used in combination with CA19.9, CUZD1 and LAMC2 were shown to significantly improve the performance of CA19.9 alone in the diagnosis of PDAC patients. We speculate that CUZD1 and LAMC2 may be good candidates to be used in a panel for monitoring PDAC patients who do not express CA19.9 levels as well as for an aid in screening high risk populations. Further validation of these two proteins is warranted.

High-throughput mass spectrometry has discovered a plethora of candidates in the biomarker field, however, subsequent verification and validation studies are urgently needed to assess the potential of novel biomarkers in the detection of pancreatic cancer. We have conducted extensive verification and validation studies on two of our most promising biomarkers CUZD1 and LAMC2 with a total of 715 blood samples. In our study, both markers demonstrated consistent diagnostic ability of early- and CA19.9 negative-PDAC cases. When used in combination with CA19.9, CUZD1 and LAMC2 were shown to significantly improve the performance of CA19.9 alone in the diagnosis of PDAC patients. We speculate that CUZD1 and LAMC2 may be good candidates to be used in a panel for monitoring PDAC patients who do not express CA19.9 levels as well as for an aid in screening high risk populations. Further validation of these two proteins is warranted.

L’adénocarcinome pancréatique canalaire s’accompagne d’une néoexpression de la mucine membranaire MUC4 et d’une surexpression des mucines membranaires MUC1 et MUC16. Ces O-glycoprotéines de haut poids moléculaire sont codées par des ARNm possédant des particularités inhabituelles par rapport aux autres transcrits humains, comme une longue demi-vie et une très grande taille. La galectine-3, une lectine endogène également surexprimée au cours du cancer pancréatique, exerce de très nombreuses fonctions biologiques, en particulier dans le domaine du trafic intracellulaire des glycoprotéines et de l’épissage des pré-ARNm. Cependant, l’implication de cette galectine à un autre niveau du cycle de vie des ARNm n’avait pas été explorée jusque-là dans la littérature. De précédents travaux du laboratoire ont démontré que la suppression de l’expression de la galectine-3 dans la lignée cellulaire cancéreuse pancréatique humaine CAPAN-1 s’accompagne d’une diminution de l’expression des transcrits de certaines mucines membranaires. L’objectif de ce travail a donc été d’étudier les mécanismes de régulation de l’expression des mucines membranaires, et plus particulièrement MUC4, par la galectine-3.Nous avons démontré que la galectine-3, in vitro, régule l’expression de MUC4 au niveau post-transcriptionnel en stabilisant les transcrits de cette mucine. Ceci passe par la potentialisation de la fixation de la RNA Binding Protein hnRNP-L sur l’élément cis-régulateur CA repeat présent dans le 3’UTR de MUC4. Nos résultats indiquent que cette régulation est présente in vivo au niveau physiologique dans des tissus épithéliaux digestifs murins. Par ailleurs, nous avons mis en évidence que la galectine-3 interagit avec hnRNP-L dans le cytoplasme mais qu’elle interagit faiblement avec des marqueurs de P-Bodies ou de granules de stress. Concernant le rôle de la galectine-3 dans le cycle de vie des ARNm, nos données révèlent que celle-ci se lie à aux transcrits matures de MUC4 au niveau périnucléaire, probablement dans des granules de stockage qui ne sont ni des granules de stress ni des P-bodies et dont le type reste à déterminer. Nous avons également élargi nos résultats en étudiant l’implication de cette lectine dans le métabolisme d’autres ARNm et nos analyses indiquent que la galectine-3 serait impliquée dans la régulation post-transcriptionnelle positive ou négative d’un ensemble de transcrits dont les fonctions convergent vers les voies UPR (Unfolded protein response) et ERAD (Endoplasmic-reticulum-associated protein degradation) mais également plus généralement vers le processing des protéines en réponse au stress du réticulum endoplasmique.En conclusion, nos travaux mettent en évidence un nouveau rôle de la galectine-3 en tant que RNA binding protein dans la stabilisation des ARNm de MUC4 mais aussi un nouveau rôle dans la coordination de l’expression de répertoires de transcrits matures ayant des rôles biologiques communs (RNA regulon) permettant à la cellule de s’adapter au plan morphologique, métabolique et biologique à des changements physiopathologiques. Ceci renforce les interconnexions largement décrites dans la littérature entre mucines, galectine-3 et les grandes fonctions cellulaires qui sont perturbées en situation cancéreuse. / Pancreatic ductal adenocarcinoma is characterized by a neo expression of the membrane-bound mucin MUC4 and an overexpression of membrane-bound mucins MUC1 and MUC16. These high molecular weight O-glycoproteins are encoded by mRNA sharing unusual features among human transcripts, such as a long half-life and a very large size. Galectin-3, an endogenous lectin frequently over-expressed in pancreatic cancer, has many biological functions, especially in intracellular glycoprotein trafficking and pre-mRNA splicing. However, the involvement of this lectin in another step of mRNA life cycle has not been explored in literature yet. Previous works performed in the laboratory have demonstrated that LGALS3 gene knock-down in a human cancerous pancreatic cancer cell line is followed by a decrease of the expression of several membrane-bound mucin mRNAs. The aim of this present work was to study the mechanism of the regulation of mucins expression, especially for MUC4, by galectin-3.We have demonstrated that galectin-3, in vitro, regulates MUC4 expression at the post-transcriptionnal level through the stabilization of the transcripts of this mucin. Galectin-3 potentiates the binding of hnRNP-L, a RNA-Binding protein, on the CA repeat region present in MUC4 3’UTR. Our results show that this regulation occurs physiologically in vivo in mice digestive epithelial tissues. Moreover, we have demonstrated that galectin-3 interacts with hnRNP-L in cell cytoplasm but scarcely with protein markers of P-Bodies or stress granules markers. Regarding the influence of galectin-3 in mRNA life cycle, our results suggest that it binds to mature MUC4 transcripts in the perinuclear area, probably in storage granules whose type should to be determined. We have also broadened our results by studying this lectin’s involvement in the metabolism of other mRNA. Our analyzes suggest that galectin-3 could be involved in the positive or negative post-transcriptionnal regulation of a mRNA subset whose functions are linked to unfolded protein response (UPR) and Endoplasmic-reticulum-associated protein degradation (ERAD) pathways, but also more generally towards protein processing in response to endoplasmic reticulum stress.In conclusion, our work highlights a new function for galectin-3 as a RNA binding protein in the stabilization of MUC4 mRNA, but also a new function in the coordination of the expression of repertories of mature transcripts with shared functions or (RNA regulon) allowing morphological, biological and metabolic cell adaptation to physiopathological changes. These results strengthen the interplay between mucins, galectin-3 and cellular functions which are disturbed in cancer.

Background: Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of US cancer related deaths. This study assessed the oncologic benefit of a neoadjuvant chemotherapy (NAC) treatment strategy for patients with clinical Stage I/II PDAC. Methods: Patients with biopsy confirmed PDAC and clinical Stage I/II disease were treated with a protocol of NAC. The primary study endpoint was median overall survival (OS). Kaplan-Meier survival curves were compared using the log-rank test. Results: 56 patients met inclusion criteria. Of these, 21 patients (38%) had Stage I disease and 35 (62%) had Stage II disease. The median OS for the entire study population was 18.7 months. A total of 22 (39%) patients were managed with NAC+S; 34 (61%) received NAC alone. Median OS and 2-year survival rates were greater in those completing NAC+S compared to NAC alone (median OS 28.8 months vs. 17.3 months: p=0.05; 2-year OS: 55% vs 21%: p=0.01) . Interestingly, patients managed with NAC who were not candidates for surgical resection after restaging demonstrated a survival advantage (17.3 months) compared to what was previously reported in historical controls. Conclusion: NAC+S provided a significant 11.5 month improvement in median OS compared to treatment with NAC alone. Modern NAC may contribute a significant oncologic benefit in the overall treatment strategy for patients with Stage I/II PDAC, even if surgery is not ultimately pursued.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Pancreatic ductal adenocarcinoma (PDAC) is an extremely deadly disease characterized by aggressive metastasis and therapeutic resistance. Reactive stroma in pancreatic tumors contributes to tumor signaling, fibrosis, inflammation, and hypoxia. Hypoxia signaling creates a more aggressive phenotype with increased potential for metastasis and decreased therapeutic efficacy. Carbonic anhydrase IX (CA9) functions as part of the cellular response to hypoxia by regulating intracellular pH to promote cell survival. Apurinic/Apyrimidinic Endonuclease-1-Reduction/oxidation Effector Factor 1 (APE1/Ref-1) is a multi-functional protein with two major activities: endonuclease activity in DNA base excision repair and a redox signaling activity that reduces oxidized transcription factors, enabling them to bind target sequences in DNA. APE1/Ref-1 is a central node in redox signaling, contributing to the activation of transcription factors involved in tumor survival, growth, and hypoxia signaling. This work evaluates the mechanisms underlying PDAC cell responses to hypoxia and APE1/Ref-1 redox signaling control of hypoxia inducible factor 1 alpha (HIF1a), a critical factor in hypoxia-induced CA9 transcription. We hypothesized that obstructing the HIF-CA9 axis at two points via APE1/Ref-1 inhibition and CA9 inhibition results in enhanced PDAC cell killing under hypoxic conditions. We found that HIF1a-mediated induction of CA9 is significantly attenuated following APE1/Ref-1 knock-down or redox signaling inhibition in patient-derived PDAC cells and pancreatic cancer-associated fibroblast cells. Additionally, dual-targeting of APE1/Ref-1 redox signaling activity and CA9 activity results in enhanced acidification and cytotoxicity of PDAC cells under hypoxic conditions as well as decreased tumor growth in an ex-vivo 3-dimensional tumor co-culture model. Further experiments characterized novel analogs of clinically relevant drugs targeting the key enzymes in this pathway, resulting in improved potency. These results underscore the notion that combination therapy is essential and demonstrate the potential clinical utility of blocking APE1/Ref-1 and CA9 function for novel PDAC therapeutic treatment.

Pancreatic ductal adenocarcinoma (PDAC) is a highly fatal cancer, accounting for 3.2% of new cancer cases yearly but nearly 8% of all yearly cancer mortalities. Over the past twenty years, our understanding of cancer biology has greatly improved which has resulted in vastly improved prognoses for many cancers. However, the prognosis of pancreatic ductal adenocarcinoma has not improved despite the advance in cancer treatments. This is especially apparent with cancer immunotherapies, a newer therapeutic strategy that utilizes the innate defense mechanism of the body to target malignancies. Immune checkpoint inhibitors are a type of cancer immunotherapy that act by inhibiting the PD-1/PD-L1 and CTLA-4 immune checkpoint pathways and allowing T lymphocytes to proliferate and generate an antitumor response. They have greatly improved the prognosis for many types of malignancies, but clinical studies show that immune checkpoint inhibition has had a limited effect on the prognosis of PDAC. Recent studies have demonstrated that the immune microenvironment of PDAC is highly immunosuppressive, which is a probable factor in limiting the therapeutic efficacy of immune checkpoint inhibitors. Myeloid derived suppressor cells (MDSCs) are a main component of the immune microenvironment in PDAC. They are immature cells of myeloid origin that express CD11b+Gr-1+ on their surface, making them phenotypically distinct from mature dendritic cells. Their infiltration of the PDAC microenvironment early on in the course of the disease is promoted in a large part by the cytokine GM-CSF. MDSCs are believed to contribute to the limited efficacy of immune checkpoint inhibitor therapy both directly and indirectly. Indirect mechanisms are mediated by promoting the activity of other immunosuppressive cells in the PDAC microenvironment such as tumor associated macrophages and regulatory T lymphocytes. MDSCs induce the transformation of naïve CD4+ T lymphocytes into protumorigenic regulatory T lymphocytes. They also promote the polarization of macrophages to the tumor associated macrophage phenotype (IL-10high IL-12low) by secreting IL-10, which decreases IL-12 synthesis by macrophages present in the tumor microenvironment. On top of mediating immunosuppression through other cell types, MDSCs directly mediate immunosuppression by decreasing the amounts of amino acids necessary for anti-tumor immunity in the tumor microenvironment and disrupting the activity of antigen presenting cells and the signaling needed to initiate a cytotoxic T lymphocyte response. The decreased amount of arginine limits the ability of T cells to proliferate, resulting in a weaker cytotoxic response. These mechanisms limit the antitumor response against pancreatic ductal adenocarcinoma, resulting in the decreased response to immune checkpoint inhibitor therapy observed in clinical trials. Future attempts to strengthen the anti-tumor immune response must be combinatorial therapies that incorporate therapeutic strategies that seek to alleviate MDSC-mediated immunosuppression of T lymphocytes from the tumor microenvironment in addition to the more widely available immune checkpoint inhibitor therapy. Such therapeutics are currently being studied in murine models and have shown promising preliminary results but have yet to have been examined in clinical trials. These therapies are an ideal avenue to explore in a search for more effective therapy for this highly lethal disease.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21420号 / 医博第4410号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 羽賀 博典, 教授 武田 俊一, 教授 坂井 義治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM