Prediction of blood-based biomarkers and subsequent design of bisulfite PCR-LDR-qPCR assay for breast cancer detection

This work is licensed under a Creative Commons Attribution 4.0 International License.Background Interrogation of site-specific CpG methylation in circulating tumor DNAs (ctDNAs) has been employed in a number of studies for early detection of breast cancer (BrCa). In many of these studies, the markers were identified based on known biology of BrCa progression, and interrogated using methyl-specific PCR (MSP), a technique involving bisulfite conversion, PCR, and qPCR. Methods In this report, we are demonstrating the development of a novel assay (Multiplex Bisulfite PCR-LDR-qPCR) which can potentially offer improvements to MSP, by integrating additional steps such as ligase detection reaction (LDR), methylated CpG target enrichment, carryover protection (use of uracil DNA glycosylase), and minimization of primer-dimer formation (use of ribose primers and RNAseH2). The assay is designed to for breast cancer-specific CpG markers identified through integrated analyses of publicly available genome-wide methylation datasets for 31 types of primary tumors (including BrCa), as well as matching normal tissues, and peripheral blood. Results Our results indicate that the PCR-LDR-qPCR assay is capable of detecting ~ 30 methylated copies of each of 3 BrCa-specific CpG markers, when mixed with excess amount unmethylated CpG markers (~ 3000 copies each), which is a reasonable approximation of BrCa ctDNA overwhelmed with peripheral blood cell-free DNA (cfDNA) when isolated from patient plasma. The bioinformatically-identified CpG markers are located in promoter regions of NR5A2 and PRKCB, and a non-coding region of chromosome 1 (upstream of EFNA3). Additional bioinformatic analyses would reveal that these methylation markers are independent of patient race and age, and positively associated with signaling pathways associated with BrCa progression (such as those related to retinoid nuclear receptor, PTEN, p53, pRB, and p27). Conclusion This report demonstrates the potential utilization of bisulfite PCR-LDR-qPCR assay, along with bioinformatically-driven biomarker discovery, in blood-based BrCa detection

環境ストレスによる人間腫瘍の分子生物学研究

筑波大学 (University of Tsukuba)201

Experimental and clinical evidence in favour of an effective immune stimulation in ER-positive, endocrine-dependent metastatic breast cancer

In ER+ breast cancer, usually seen as the low immunogenic type, the main mechanisms favouring the immune response or tumour growth and immune evasion in the tumour microenvironment (TME) have been examined. The principal implications of targeting the oestrogen-mediated pathways were also considered. Recent experimental findings point out that anti-oestrogens contribute to the reversion of the immunosuppressive TME. Moreover, some preliminary clinical data with the hormone-immunotherapy association in a metastatic setting support the notion that the reversion of immune suppression in TME is likely favoured by the G0-G1 state induced by anti-oestrogens. Following immune stimulation, the reverted immune suppression allows the boosting of the effector cells of the innate and adaptive immune response. This suggests that ER+ breast cancer is a molecular subtype where a successful active immune manipulation can be attained. If this is confirmed by a prospective multicentre trial, which is expected in light of the provided evidence, the proposed hormone immunotherapy can also be tested in the adjuvant setting. Furthermore, the different rationale suggests a synergistic activity of our proposed immunotherapy with the currently recommended regimen consisting of antioestrogens combined with cyclin kinase inhibitors. Overall, this lays the foundation for a shift in clinical practice within this most prevalent molecular subtype of breast cancer

종양 미세환경에서 대식세포 헴 산화 효소의 역할

학위논문 (박사)-- 서울대학교 대학원 : 의과대학 협동과정 종양생물학전공, 2019. 2. 서영준.One of the most common treatment options for breast cancer is chemotherapy. After chemotherapy, however, unwanted host effects provoke tumor recurrence and aggressiveness of cancer cells which often arise as a consequence of disruption in the patients immune system. Lymphocytes, such as CD8+ cytotoxic T cells which have capability of suppressing cancer progression, are depleted following chemotherapy. Tumor-associated macrophages (TAMs), an abundant set of tumor-infiltrating myeloid cells in tumor microenvironment, play an important role in immunosuppression which often occurs during conventional chemotherapy. Dying cancer cells generated during the chemotherapy can potentially hijack accumulated TAMs, provoking tumor recurrence. Therefore, reprogramming of TAMs to maximize the chemotherapeutic efficacy is considered a promising novel anticancer strategy. In this study, I investigated whether tumor cell debris generated as a consequence chemotherapy can reduce therapeutic efficacy by modulating the activity of tumor-infiltrating macrophages. In a 4T1 syngeneic murine breast cancer model, the expression of the M1 marker, CD86 in the TAMs and the infiltration of CD8+ T cells was reduced following paclitaxel (PTX) treatment. PTX treatment also resulted in an enhancement of heme oxygenase-1 (HO-1) expression in tumor-infiltrating myeloid cells engulfing tumor cell debris. Consistent with the in vivo profile of TAMs, bone marrow-derived macrophages (BMDMs) phagocytosing breast tumor cell debris exhibited significant upregulation of HO-1 expression. HO-1 induction in BMDMs engulfing breast tumor cell debris inhibited M1 polarization and reprogramed macrophages to the M2 phenotype. In contrast, inhibition of HO-1 activity with zinc protoporphyin IX resulted in sustained M1 macrophage activity of BMDMs co-cultured with breast cancer cell debris. Therapeutic efficacy of PTX to suppress the tumor growth was significantly enhanced in HO-1 knock out mice bearing 4T1 breast cancer. Consistent with that finding, pharmacologic inhibition of HO-1 activity augmented the therapeutic efficacy of PTX by stimulating CD86+ M1 TAMs in a 4T1 breast cancer. Furthermore, blockade of HO-1 in breast tumor bearing mice promoted CD8+ T cell infiltration and activity. Taken together, the above findings suggest that tumor cell debris-induced HO-1 overexpression in macrophages during chemotherapy dampens therapeutic efficacy by manipulating anti-tumor immunity.유방암 치료를 위하여 많이 쓰이는 치료방법 중 하나는 화학요법이다. 그러나 화학 요법 치료 후 면역 시스템의 문제로 암의 재발이나 악성화가 진행 되기도 한다. 특히, 암 진행을 억제하는 중요한 역할을 가지고 있는 CD8+ T세포가 화학 요법 후 활성이 떨어지기도 한다. 종양 미세환경에서 많은 부분을 차지하고 있는 종양 관련 대식세포는 화학 요법 치료에서 항암 면역 반응을 조절하는 역할을 가지고 있다. 화학 요법으로 인하여 생성된 사멸된 암세포는 종양 관련 대식세포를 조절 하여 암을 재발시키기도 한다. 그러므로 종양 관련 대식세포를 재프로그래밍 하여서 화학요법의 효과를 극대화시키는 방법이 항암 치료 전략 중 주목받고 있다. 이번 연구에서는 화학 요법 후 사멸된 암세포가 대식세포의 활성을 조절하여 항암 치료의 효과를 줄이는지 알아보았다. 4T1 유방암 모델에서 파클리탁셀을 처리한 군에서는 CD86+M1 대식 세포와 CD8+ T세포의 비율이 줄어들었다. 흥미롭게도 파클리탁셀을 투여한 군에서 사멸된 암세포를 잡아먹은 대식세포의 헴산화 효소 발현이 증가 되어있었다. 또한, 사멸된 암세포를 잡아먹은 골수 유래 대식 세포의 헴 산화 효소 발현도 확인 할 수 있었다. 사멸된 암세포를 잡아먹은 골수 유래 대식세포의 헴 산화 효소의 발현은 M1 극성화를 줄이고 M2 극성화를 증가 시켰다. 반대로 헴 산화 효소를 비활성화 시키면 사멸된 암세포를 잡아 먹은 골수 유래 대식 세포의 M1 활성이 유지됨을 관찰할 수 있었다. 헴 산화 효소 녹아웃 마우스를 사용한 4T1 유방암 모델에서는 파클리탁셀의 치료 효과가 증진되었다. 마찬가지로, 4T1 유방암 모델에서 zinc protoporphyin IX를 사용하여 HO-1 활성을 억제하니 CD86+ M1 대식 세포 비율이 증진되어 파클리탁셀의 치료 효과를 증가시켰다. 또한 유방암 모델에서 헴 산화 효소의 억제는 CD8+ T세포의 유입을 증진시켰다. 이를 통해, 화학요법으로 생성된 사멸된 암세포를 통하여 대식 세포의 증가된 헴 산화 효소는 항암 면역을 억제하여 항암치료 효과를 감소 시킨다는 것을 알았다. 이러한 결과는 유방암 종양 미세 환경에서 화학요법으로 증가된 헴 산화 효소를 표적으로 치료 하면 면역 시스템을 조절하여 화학 요법을 증진시킬 수 있음을 시사한다.TABLE OF CONTENTS ABSTRACT----------------------------------------------------------------------i TABLE OF CONTENTS---------------------------------------------------iv LIST OF FIGURES-----------------------------------------------------------ix LIST OF ABBREVIATIONS--------------------------------------------xiii 1. Introduction-----------------------------------------------------------------------1 2. Materials and Methods-----------------------------------------------------------5 3. Results ---------------------------------------------------------------------------16 3.1 Chemotherapy induces an immunosuppressive TME in breast cancer-------------------------------------------------------------------------------------------16 3.2 Phagocytosis of tumor cell debris regulates the polarization of macrophages to a pro-tumor phenotype-----------------------------------------17 3.3 Engulfment of tumor cell debris induces HO-1 expression in macrophages-----------------------------------------------------------------------20 3.4 HO-1 overexpression triggered by phagocytosis of tumor cell debris regulates the polarization of macrophages--------------------------------------21 3.5 HO-1 inactivation amplifies the therapeutic efficacy of PTX ----------23 3.6 HO-1 inhibition promotes anti-tumor T cell function in response to PTX treatment---------------------------------------------------------------------------24 3.7 HO-1 inactivation-induced M1 TAMs are crucial for the enhanced response to PTX therapy----------------------------------------------------------25 4. Discussion-----------------------------------------------------------------------69 5. References-----------------------------------------------------------------------75 Appendix---------------------------------------------------------------------------90 Taurine chloramine potentiates phagocytic activity of peritoneal macrophages through upregulation of dectin-1 mediated by heme oxygenase-1-derived carbon monoxide 1. Abstract--------------------------------------------------------------------------92 2. Introduction----------------------------------------------------------------------94 3. Materials and Methods---------------------------------------------------------97 4. Results --------------------------------------------------------------------------104 4.1 TauCl potentiates host defense to fungal infection-----------------------104 4.2 TauCl promotes phagocytic efficiency of peritoneal macrophages in a fungal infection-------------------------------------------------------------------104 4.3 TauCl increases dectin-1 expression in macrophages of mice infected with fungal pathogens-----------------------------------------------------------106 4.4 TauCl-induced HO-1 expression is critical for upregulation of dectin-1 expression in macrophages in a murine peritonitis---------------------------107 4.5 TauCl-induced HO-1 expression is essential for enhanced phagocytic activity of macrophages in a murine peritonitis-------------------------------108 4.6 CO enhances phagocytic activity of murine macrophages through upregulation of dectin-1 expression--------------------------------------------109 4.7 TauCl-induced HO-1 expression upregulates dectin-1 expression through PPAR-γ activation------------------------------------------------------109 5. Discussion----------------------------------------------------------------------134 6. References----------------------------------------------------------------------140 ABSTRACT IN KOREAN (국문초록) --------------------------------------148Docto

Υποδοχείς Farnesoid X Reseptors (FXRs) και καρκίνος προστάτη

Σκοπός της παρούσας βιβλιογραφικής ανασκόπησης ήταν η μελέτη του παρόντος δημοσιευμένου υλικού επάνω στη συσχέτιση του πυρηνικού υποδοχέα χολικών οξέων FXR με τη δημιουργία και εξέλιξη του καρκίνου του προστάτη. Ο διαρκώς αυξανόμενος όγκος ερευνών τονίζει τη σημασία του εν λόγω υποδοχέα στο μεταβολισμό και συγκεκριμένα στη διαχείριση των λιπιδίων σε συνδυασμό με τον παγιωμένο ρόλο των ανδρογόνων και των υποδοχέων τους στην ογκογένεση του καρκίνου του προστάτη, αλλά και η ήδη εξακριβωμένη εμπλοκή του με κακοήθεις νεοπλασίες άλλων οργάνων, όπως του μαστού, τον καθιστούν ως ελκυστικό πεδίο έρευνας και στόχο ανάπτυξης νέων φαρμάκων και θεραπευτικών επιλογών. Η ανασκόπηση έφτασε στο συμπέρασμα ότι, παρά τα πολύ υποσχόμενα υπάρχοντα δεδομένα, δυστυχώς, δεν υπάρχουν επαρκή στοιχεία ακόμα για ασφαλή συμπεράσματα. Επομένως, καθίσταται υψίστης σημασίας ο σχεδιασμός περισσότερων ερευνών οι οποίες να περιλαμβάνουν και ζωικά μοντέλα για τη διεξαγωγή ασφαλέστερων συμπερασμάτων σχετικά με αυτό το πολλά υποσχόμενο αντικείμενο.The purpose of this literature review was to investigate current published scientific data on the relation of bile acid nuclear receptor FXR and prostate cancer development and evolution. Constantly announced evidence on the importance of FXR on metabolism and especially on lipid control combined with the certain role of androgens and their receptors on prostate tumourigenesis, as well as already known involution of FXR and other cancers like breast, provide an appealing research field and a promising pharmacological target and therapeutic option. This review concluded that, unfortunately, there is not enough evidence yet on this matter, although the first results are very encouraging, rendering further research publications, including animal models, of paramount significance

Caractérisation des partenaires d'interaction du récepteur LRH-1 (Liver Receptor Homolog 1) dans un modèle cellulaire de cancer du sein triple négatif

Le cancer du sein représente le cancer le plus souvent diagnostiqué chez les femmes à travers le monde. Un sous-type moléculaire de cancer du sein, dit triple négatif (TN), regroupe un ensemble de tumeurs caractérisées par l’absence d’expression des récepteurs aux oestrogènes (ER), à la progestérone (PR) ainsi que d’un récepteur de facteur de croissance épidermique (HER2). Ces tumeurs, pour lesquelles les options thérapeutiques sont plus limitées, faute de cibles spécifiques, représentent le sous-type de cancer du sein pour lequel le pronostic est le plus sombre. Ces cellules sont caractérisées par une prolifération cellulaire marquée et une grande capacité de migration et d’invasion propices à la formation rapide de métastases, des phénotypes qui ont été associés à une protéine appelée Liver receptor homolog 1 (LRH-1). LRH-1 agit comme facteur de transcription permettant de réguler l’expression d’un vaste ensemble de gènes, et son activité transcriptionnelle est très spécifique aux tissus, où il semble interagir avec des cofacteurs variables. LRH-1 semble opérer un programme d’expression génique distinct dans les cellules TN par rapport aux autres sous-types de cancers du sein. L’hypothèse de ce projet de recherche est que LRH-1 interagit avec des cofacteurs spécifiques aux cellules TN pouvant agir à titre de coactivateurs ou de corépresseurs. L’objectif du projet était donc d’identifier les cofacteurs potentiels de LRH-1 à la chromatine à l’aide de deux approches actuelles en protéomique, le BioID et le RIME, toutes deux réalisées dans des lignées cellulaires humaines dérivées de tumeurs TN (MDA-MB-231). Le BioID n’a pas permis d’identifier de cofacteurs potentiels puisque la fusion de LRH-1 avec BirA* en C-terminal a semblé affecter le fonctionnement de LRH-1, entraînant sa translocation au cytoplasme, alors qu’en présence d’une fusion en N-terminal la biotinylation de partenaires potentiels n’a pu être observée à un niveau d’induction de la protéine de fusion similaire au niveau d’expression endogène de LRH-1. Le RIME a permis de générer une liste de 150 cofacteurs potentiels de LRH-1, parmi lesquels RUNX2, CBFB, GSK3B et ARRB2 retiennent particulièrement l’attention pour leur rôle déjà décrit dans un contexte de progression tumorale. Toutefois, d’autres expérimentations sont nécessaires pour confirmer l’interaction entre ces facteurs et LRH-1, de même que l’implication potentielle de cette interaction sur l’expression d’un transcriptome propre aux cellules TN