Cancer associated fibroblasts: the architects of stroma remodelling

Fibroblasts have exceptional phenotypic plasticity and capability to secrete vast amount of soluble factors, ECM components and extracellular vesicles. While in physiological conditions this makes fibroblasts master regulators of tissue homeostasis and healing of injured tissues, in solid tumours cancer-associated fibroblasts (CAFs) co-evolve with the disease, and alter the biochemical and physical structure of the tumour microenvironment, as well as the behaviour of the surrounding stromal and cancer cells. Thus CAFs are fundamental regulators of tumour progression and influence response to therapeutic treatments. Increasing efforts are devoted to better understand the biology of CAFs to bring insights to develop complementary strategies to target this cell type in cancer. Here we highlight components of the tumour microenvironment that play key roles in cancer progression and invasion, and provide an extensive overview of past and emerging understanding of CAF biology as well as the contribution that mass spectrometry (MS)-based proteomics has made to this field

Integrated Proteotranscriptomics of Breast Cancer Reveals Globally Increased Protein-mRNA Concordance Associated with Subtypes and Survival

BACKGROUND: Transcriptome analysis of breast cancer discovered distinct disease subtypes of clinical significance. However, it remains a challenge to define disease biology solely based on gene expression because tumor biology is often the result of protein function. Here, we measured global proteome and transcriptome expression in human breast tumors and adjacent non-cancerous tissue and performed an integrated proteotranscriptomic analysis. METHODS: We applied a quantitative liquid chromatography/mass spectrometry-based proteome analysis using an untargeted approach and analyzed protein extracts from 65 breast tumors and 53 adjacent non-cancerous tissues. Additional gene expression data from Affymetrix Gene Chip Human Gene ST Arrays were available for 59 tumors and 38 non-cancerous tissues in our study. We then applied an integrated analysis of the proteomic and transcriptomic data to examine relationships between them, disease characteristics, and patient survival. Findings were validated in a second dataset using proteome and transcriptome data from The Cancer Genome Atlas and the Clinical Proteomic Tumor Analysis Consortium. RESULTS: We found that the proteome describes differences between cancerous and non-cancerous tissues that are not revealed by the transcriptome. The proteome, but not the transcriptome, revealed an activation of infection-related signal pathways in basal-like and triple-negative tumors. We also observed that proteins rather than mRNAs are increased in tumors and show that this observation could be related to shortening of the 3\u27 untranslated region of mRNAs in tumors. The integrated analysis of the two technologies further revealed a global increase in protein-mRNA concordance in tumors. Highly correlated protein-gene pairs were enriched in protein processing and disease metabolic pathways. The increased concordance between transcript and protein levels was additionally associated with aggressive disease, including basal-like/triple-negative tumors, and decreased patient survival. We also uncovered a strong positive association between protein-mRNA concordance and proliferation of tumors. Finally, we observed that protein expression profiles co-segregate with a Myc activation signature and separate breast tumors into two subgroups with different survival outcomes. CONCLUSIONS: Our study provides new insights into the relationship between protein and mRNA expression in breast cancer and shows that an integrated analysis of the proteome and transcriptome has the potential of uncovering novel disease characteristics

Cellular interactions in the tumor microenvironment: the role of secretome

Over the past years, it has become evident that cancer initiation and progression depends on several components of the tumor microenvironment, including inflammatory and immune cells, fibroblasts, endothelial cells, adipocytes, and extracellular matrix. These components of the tumor microenvironment and the neoplastic cells interact with each other providing pro and antitumor signals. The tumor-stroma communication occurs directly between cells or via a variety of molecules secreted, such as growth factors, cytokines, chemokines and microRNAs. This secretome, which derives not only from tumor cells but also from cancer-associated stromal cells, is an important source of key regulators of the tumorigenic process. Their screening and characterization could provide useful biomarkers to improve cancer diagnosis, prognosis, and monitoring of treatment responses.Agência financiadora Fundação de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) FAPESP 10/51168-0 12/06048-2 13/03839-1 National Council for Scientific and Technological Development (CNPq) CNPq 306216/2010-8 Fundacao para a Ciencia e a Tecnologia (FCT) UID/BIM/04773/2013 CBMR 1334info:eu-repo/semantics/publishedVersio

New protein clustering of breast cancer tissue proteomics using actin content as a cellularity indicator

In the present study, we report the comparative proteome profiles of proteins solubilized from 37 breast cancer surgical tissues, normalized for the actin content. Blood-derived proteins were excluded from the analysis. Among the tumor-derived protein spots, a large proportion (39%) was found present in all patients. These included several glycolytic enzymes, detox and heat shock proteins, members of annexin and S100 protein families, cathepsin D, and two "rare" proteins, DDAH2 involved in the angiogenesis control, and the oncogene PARK7. Other proteins, such as psoriasin, galectin1, cofilin, peroredoxins, SH3L1, and others, showed sporadic presence and high expression level, which suggests their possible role for patient stratification

Disease proteomics

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62680/1/nature01514.pd

오믹스 데이터를 이용한 개와 사람의 바이오마커 비교연구

학위논문 (박사) -- 서울대학교 대학원 : 수의과대학 수의학과, 2021. 2. 조제열.Breast cancer (BC), known as mammary gland carcinoma (MGC), is one of the most frequently diagnosed malignancies among women and canines. Despite the countless efforts to fully understand and overcome such cancer-related anomalies, various subtypes originating from specific regions of the mammary organ generates infrequent yet menacing malignancies. Comparative medicinal approach has emerged as a powerful method to approach human BC research on a different perspective. Together with various omics technologies, the paradigm for BC treatment has become shifting toward evidence-based large-scale discovery studies which leads to biomarkers specifically expressed in distinct BC subtypes. The incorporation of diverse omics data spreading from next generation sequencing (NGS) assembled epigenetic transcripts to mass spectrometry (MS) derived proteomics stands as a solution for breast malignancy differential diagnosis and drug target discovery. The research is divided into three chapters for detailed description. CHAPTER Ⅰ describes sequenced RNA-seq data from ten pairs of canine mammary gland carcinoma (MGC) and matching adjacent normal tissues to identify canine MGC-associated transcriptomic signatures. Breast cancer (BC) and MGC is the most frequently diagnosed and leading cause of cancer-related mortality in both women and canines. To better understand both canine MGC- and human BC-specific genes which express similar transcriptomic profiles, we sequenced RNAs obtained from eight pairs of carcinomas and adjacent normal tissues in dogs. By comprehensive transcriptome analysis, 351 differentially expressed genes (DEGs) were identified in overall canine MGCs. Based on the DEGs, comparative analysis revealed correlation existing among the three histological subtypes of canine MGC (ductal, simple, and complex) and four molecular subtypes of human BC (HER2+, ER+, ER & HER2+, and TNBC). Eight DEGs shared by all three subtypes of canine MGCs had been previously reported as cancer-associated genes in human studies. Gene ontology (GO) and pathway analyses using the identified DEGs revealed that the biological processes of cell proliferation, adhesion, and inflammatory responses are enriched in up-regulated MGC DEGs. In contrast, fatty acid homeostasis and transcription regulation involved in cell fate commitment were down-regulated in MGC DEGs. Moreover, correlations are demonstrated between upstream promoter transcripts and DEGs. Canine MGC- and subtype-enriched gene expression allows us to better understand both human BC and canine MGC, yielding new insight into the development of biomarkers and targets for both diseases. The resemblance in transcriptomic profiles will present canines as a suitable comparative model for MGC studies and its application to human BC. CHAPTER Ⅱ focuses on the identification and treatment specific to a BC subtype. Among many types of BCs, triple-negative breast cancer (TNBC) has the worst prognosis and the least cases reported. To gain a better understanding and a more decisive precursor for TNBC, two major histone modifications, an activating modification H3K4me3 and a repressive modification H3K27me3, were analyzed using data from normal breast cell lines against TNBC cell lines. The combination of these two histone markers on the gene promoter regions showed a great correlation with gene expression. A list of signature genes was defined as active (highly enriched H3K4me3), including NOVA1, NAT8L, and MMP16, and repressive genes (highly enriched H3K27me3), IRX2 and ADRB2, according to the distribution of these histone modifications on the promoter regions. To further enhance the investigation, potential candidates were also compared with other types of BC to identify signs specific to TNBC. RNA-seq data was implemented to confirm and verify gene regulation governed by the histone modifications. Combinations of the biomarkers based on H3K4me3 and H3K27me3 showed the diagnostic value area under the curve (AUC) 93.28% with P-value of 1.16e-226. The results of this study suggest that histone modification analysis of opposing histone modifications may be valuable toward developing biomarkers and targets for TNBC and further provide understanding the overall regulation derived by epigenetic modifications. CHAPTER Ⅲ consists of biomarker study implemented from canine mammary tumors to human BCs. While biomarkers are continuously discovered, specific markers representing the aggressiveness and invasiveness of BC are lacking compared to classification markers. In this study, samples from canine mammary tumors were used in a comparative approach. An extensive 36 fractions of both canine normal and MGC plasma was subjected to high-performance quantitative proteomics analysis. Among the identified proteins, Lecithin-Cholesterol Acyltransferase (LCAT) was discovered to be selectively expressed in mixed tumor samples, which represents an aggressive developed stage of cancer, possibly highly metastatic. With further multiple reaction monitoring (MRM) and western blot validation, we discovered that the LCAT protein is an indicator of aggressive mammary tumor. Interestingly, we also found that LCAT is overexpressed in high grade and lymph node positive BC in silico data. We also demonstrated that LCAT is highly expressed in the sera of advanced stage human BCs within the same classification. In conclusion, we identified a possible common plasma protein biomarker, LCAT, that is highly expressed in aggressive human BC and canine mammary tumor.유방암은 여성과 암캐에서 가장 빈번하게 진단되는 악성종양 중 하나이다. 이러한 암과 관련된 이상현상을 완전히 이해하고 극복하려는 수많은 노력에도 불구하고, 유방 조직의 특정 부위에서 발생하는 여러 유형들은 드물지만 위협적인 악성 종양으로 발달한다. 비교 의학적 접근법은 인간의 유방암 연구에 기존과는 다른 관점으로 접근하는 효과적인 방법으로 등장했다. 다양한 오믹스 기술의 등장과 함께 유방암 치료의 전반적인 방향이 대규모 데이터를 이용하여 특정 유방암을 지칭하는 바이오마커 발굴로 기울었다. 차세대 염기서열 분석(NGS)을 이용한 후생유전체 데이터부터 질량분석기(MS)에서 생산하는 단백체 정보까지, 이러한 오믹스 데이터를 통합분석하는 것이 악성 유방암 진단과 약물 표적 발견을 위한 해결책이다. 본 연구는 총 3장으로 구성된다. 제1장에서는 10쌍의 개 유선암 및 인접 정상 조직에서 추출한 RNA-seq 데이터로 개 유선암과 연관된 신호를 식별하는 방법을 설명한다. 유방암(BC)/유선암(MGC)은 가장 빈번한 암중 하나이며 암과 관련된 사망률에서 선두를 차지하고 있다. 개 유선암과 사람 유방암 특이적 유전자를 이해하기 위해, 우리는 개의 8쌍의 발암과 인접한 정상 조직에서 얻은 RNA의 염기서열을 분석했다. 전사체 분석을 통해 개 전체 유선암에서 351개의 특이적 발현유전자를 확인했다. 비교분석 결과, 개 유선암의 세 가지 조직학적 유형(단순형, 관상형, 복합형)과 인간 유방암의 네 가지 분자 유형(HER2+, ER+, ER&HER2+, TNBC) 사이에 존재하는 상관관계를 밝혔다. 세 종류의 개 유선암을 모두 공유하는 8개의 DEG는 이전에 인간 연구에서 암과 관련된 유전자로 보고됬다. 확인된 DEG를 이용한 유전자 온톨로지 및 발현 경로 분석 결과, 세포 증식, 접착, 염증 반응 과정이 유선암 DEG에서 나타났다. 이와는 대조적으로, 세포 사멸과 관련된 전사체 조절 및 지방산 항상성에 연관된 유선암 DEG들은 하향 조절되었다. 더욱이, 상류 프로모터 전사체(PROMPT)와 DEG 사이에 상관관계가 있음을 밝혔다. 개 유선암 및 조직학적 유형 특이적 발현 유전자를 통해 우리는 인간의 유방암과 개 유선암을 더 잘 이해할 수 있게 되었으며, 두 질병의 바이오마커의 진단과 개발에 대한 새로운 통찰력을 얻을 수 있을 것이다. 제2장은 특정 유방암 유형의 판별과 치료에 초점을 맞추고있다. 여러 유방암 유형 중 삼중음성유방암(TNBC)은 예후가 가장 나쁘며 보고된 사례가 가장 적다. TNBC에 대한 보다 나은 이해와 효과적인 전구체을 얻기 위해 TNBC 세포와 정상 유방 세포의 데이터를 사용하여 두 가지 주요 히스톤 변형인 활성화 변형체 H3K4me3와 억압 변형체 H3K27me3를 분석하였다. 프로모터 유전자에 두 히스톤 변형체의 조합을 통해 유전자 발현과 높은 상관관계가 있음을 확인했다. 유전자의 목록은 NOVA1, NAT8L, MMP16을 포함한 활성화된 유전자(H3K4me3이 많이 포진된)와 IRX2, ADRB2와 같은 억제된 유전자(H3K27me3이 많이 포진된)로 정의됐다. 추가적인 조사를 위해, 후보 유전자들은 TNBC에 특이적인 발현함을 식별하기 위해 다른 종류의 유방암과 비교했다. RNA-seq 데이터는 히스톤 변형에 의해 지배되는 유전자 조절을 확인하고 검증하기 위해 구현됐다. H3K4me3와 H3K27me3를 통합하여 분석한 바이오마커 조합은 P-값이 1.16e-226인 AUC 93.28%를 보였다. 이 연구 결과는 프로모터 지역에 위치한 서로 반대되는 히스톤 변형 분석이 TNBC의 바이오마커의 진단 및 개발에 활용될 수 있음을 시사하며 발현의 과정이 후성유전체에 의한 조절 기작과 관련되어 있기에 이러한 유전자 발현에 대한 연구방향을 제시해 줄 수 있을 것이다. 제3장은 개 유선암에서 시작하여 인간 유방암까지 적용될 수 있는 바이오마커 연구로 구성된다. 바이오마커는 지속적으로 발견되지만, 유방암의 공격성과 지속성을 대표해주는 바이오마커는 유방암의 유형을 분류시키는 바이오마커에 비해 부족하다. 이 연구는 비교의학적 접근법을 통한 개 유선 종양 샘플을 사용했다. 개암 정상 혈장과 유선암 혈장 모두 36분할을 통한 광범위한 정량적 단백체 분석을 진행했다. 확인된 단백질 중 LCAT는 전이 가능성이 높은 공격적인 암 발병 단계를 나타내는 혼합형 종양 검체에서 특이적으로 발현되는 것으로 밝혀졌다. 추가적인 질량분석과 Western Blot 검증을 통해 우리는 LCAT 단백질이 전이성이 높은 유선종양의 지표단백질이 될 수 있음을 발견했다. 흥미롭게도, 사람의 림프절 양성 유방암에서 과발현된 LCAT이 환자의 수명을 유의미하게 줄이며 유방암 중 2기 이상 진행되었을 때에도 개 유선암과 동일하게 높게 발현되는 것을 확인하였다. 이것으로 단백질 LCAT은 사람과 개에서 공격적인 형태의 유방암 및 유선암을 지칭하는 지표단백질로서의 가능성을 밝혔다.ABSTRACT i CONTENTS v LIST OF FIGURES viii LIST OF TABLES x ABBREVIATIONS xi BACKGROUND 1 1. BREAST CANCER 1 2. COMPARATIVE MEDICINE 6 3. BIOMARKERS 11 4. NEXT GENERATION SEQUENCING 14 5. MS BASED PROTEOMICS 18 CHAPTER Ⅰ Transcriptome Signatures of Canine Mammary Gland Carainomas and Its Comparison to Human Breast Cancers 22 1. INTRODUCTION 23 2. MATERIALS AND METHODS 27 3. RESULTS 32 4. DISCUSSION 60 CHAPTER Ⅱ Analysis of Opposing Histone Modifications H3K4me3 and H3K27me3 Reveals Candidate Diagnostic Biomarkers for TNBC and Gene Set Prediction Combination 67 1. INTRODUCTION 68 2. MATERIALS AND METHODS 71 3. RESULTS 76 4. DISCUSSION 87 CHAPTER Ⅲ Common Plasma Protein Marker LCAT in Aggressive Human Breast Cancer and Canine Mammary Gland Carcinoma. 91 1. INTRODUCTION 92 2. MATERIALS AND METHODS 94 3. RESULTS 97 4. DISCUSSION 108 GENERAL DISCUSSION 112 GENERAL CONCLUSION 115 REFERENCES 117 ABSTRACT IN KOREAN 132Docto

PROTEOMIC IDENTIFICATION OF NOVEL MARKERS IN BREAST AND COLON CANCER

Background: Discovery of new biomarker represent the greatest promise for the detection and management of cancer. Although progress in cancer biology has been rapid during the past few years, the complete understanding of molecular basis for cancer initiation, progression and efficacious treatments is still lacking. In this context, the application of proteomic strategies is now holding a focal position. The main reason is that proteins are the functional players that drive cancer phenotypes. Among cancers, breast and colon represent the most frequent forms. The evolution of these type of cancer are not easily predictable since there are several types that behave differently among patients. The biological heterogeneity is consistent with observed varied responses to therapies across patients, also. On the other hand, drug delivery is an emergent field focused on targeting drugs to a desirable group of cells, in order to minimize undesirable side-effects and maximize the therapeutic activity. Metallic nanoparticles, in particular silver nanoparticles (Ag-NPs) exhibit low toxicity to mammalian cells (Mahapatra and Karak, 2008) and are good candidate as smart therapeutics. Based on these evidences, the first part of the study was aimed to discover new potential protein biomarkers in breast and colon cancer tissues and sera, using proteomic techniques, useful as diagnostic and prognostic factors in vivo. The second part of the study was focused on the in vitro cytotoxic effects of silver nanoparticles Ag-NPs embedded on Klebsiella Oxytoca DSM29614 (KO) Exopolysaccaride (EPS), produced in aerobic versus anaerobic conditions. Methods: Diagnostic biomarkers in breast and colon cancer: Taken advantage from previous results by the proteomic analysis performed on 13 breast cancer tissues and their matched non-tumoral adjacent tissues (Pucci-Minafra et al., 2007), we first analyze by 2D-DIGE pool of both breast and colon cancer tissues extracts compared to the matched pool of non tumoral adjacent tissues extracts. Differentially expressed proteins, identified by Maldi-TOF/TOF, were functionally clustered. We also investigate the activity levels of MMP-2 and MMP-9 in breast and colon tissues as well as in sera of the same patients. Prognostic biomarkers in breast and colon cancer: In order to identity putative proteomic signatures for colorectal cancer (CRC) metastasis, a comparative profiling of a colon cancer tissue paired with the non tumoral adjacent mucosa and with the liver metastasis from the same patient was performed. A three-step approach (normal versus tumoral versus metastasis) was used to select unique proteins involved in liver metastasis. For breast cancer, a large proteomic investigation performed on a large sample set of breast cancer patients (Cancemi et al., 2010, 2012), pointed the important role of S100 protein members in breast cancer progression. Using on line tools, for instance GOBO and breast cancer Kaplan Meir-plotter we assessed gene expression levels and clinical correlations of S100 proteins in breast patients. Cytotoxic effects of silver nanoparticles biosynthesized from KO (Ag-NPs-EPS) in SK-BR3 breast cancer cell line: We monitored cell proliferation inhibition rate by MTT assay, morphological changes and proteomic modulation. Results: Diagnostic biomarkers in breast and colon cancer: Differentially breast and bolon proteomic profiling revealed several proteins involved in common pathways among the type of cancer. The important role of MMPs in tumorigenesis was confirmed by our observations regarding their major expressions in cancer tissues compared to the normal tissues. Prognostic biomarkers in breast and colon cancer: Among the differentially expressed proteins between normal-tumor and liver metastasis, Cathepsin D expression was further analyzed as prognostic factor in CRC. Moreover, integrating results obtained by bioinformatics analysis performed on breast cancer gene expression dataset confirmed the important role of S100 proteins in breast cancer progression. Cytotoxic effects of silver nanoparticles (AgNPs) biosynthesized from KO in SK-BR3 breast cancer cell line: The most important effects were obtained by aerobically AgNPs-EPS treatment, due to the major release of Ag+1, as verified by voltammetry analysis. Morphological alteration were consistent with apoptotic features. Proteomic analysis showed modulation of several proteins related to oxidative stress and apoptotic and mitochondrial pathways. Conclusions: Conclusively, the present study contribute to the implementation of the panel of new proteomic biomarkers useful for diagnostic and prognostic applications in breast and colon cancer, providing new informations about the effects of the biosynthesized Ag-NPs-EPS on breast cancer cells

Delineation of pathogenomic insights of breast cancer in young women

© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).The prognosis of breast cancer (BC) in young women (BCYW) aged ≤40 years tends to be poorer than that in older patients due to aggressive phenotypes, late diagnosis, distinct biologic, and poorly understood genomic features of BCYW. Considering the estimated predisposition of only approximately 15% of the BC population to BC-promoting genes, the underlying reasons for an increased occurrence of BCYW, at large, cannot be completely explained based on general risk factors for BC. This underscores the need for the development of next-generation of tissue- and body fluid-based prognostic and predictive biomarkers for BCYW. Here, we identified the genes associated with BCYW with a particular focus on the age, intrinsic BC subtypes, matched normal or normal breast tissues, and BC laterality. In young women with BC, we observed dysregulation of age-associated cancer-relevant gene sets in both cancer and normal breast tissues, sub-sets of which substantially affected the overall survival (OS) or relapse-free survival (RFS) of patients with BC and exhibited statically significant correlations with several gene modules associated with cellular processes such as the stroma, immune responses, mitotic progression, early response, and steroid responses. For example, high expression of COL1A2, COL5A2, COL5A1, NPY1R, and KIAA1644 mRNAs in the BC and normal breast tissues from young women correlated with a substantial reduction in the OS and RFS of BC patients with increased levels of these exemplified genes. Many of the genes upregulated in BCYW were overexpressed or underexpressed in normal breast tissues, which might provide clues regarding the potential involvement of such genes in the development of BC later in life. Many of BCYW-associated gene products were also found in the extracellular microvesicles/exosomes secreted from breast and other cancer cell-types as well as in body fluids such as urine, saliva, breast milk, and plasma, raising the possibility of using such approaches in the development of non-invasive, predictive and prognostic biomarkers. In conclusion, the findings of this study delineated the pathogenomics of BCYW, providing clues for future exploration of the potential predictive and prognostic importance of candidate BCYW molecules and research strategies as well as a rationale to undertake a prospective clinical study to examine some of testable hypotheses presented here. In addition, the results presented here provide a framework to bring out the importance of geographical disparities, to overcome the current bottlenecks in BCYW, and to make the next quantum leap for sporadic BCYW research and treatment.info:eu-repo/semantics/publishedVersio

Syntenin-1 is a promoter and prognostic marker of head and neck squamous cell carcinoma invasion and metastasis.

Metastasis represents a key factor associated with poor prognosis of head and neck squamous cell carcinoma (HNSC). However, the underlying molecular mechanisms remain largely unknown. In this study, our liquid chromatography with tandem mass spectrometry analysis revealed a number of significantly differentially expressed membrane/membrane-associated proteins between high invasive UM1 and low invasive UM2 cells. One of the identified membrane proteins, Syntenin-1, was remarkably up-regulated in HNSC tissues and cell lines when compared to the controls, and also over-expressed in recurrent HNSC and high invasive UM1 cells. Syntenin-1 over-expression was found to be significantly associated with lymph node metastasis and disease recurrence. HNSC patients with higher syntenin-1 expression had significantly poorer long term overall survival and similar results were found in many other types of cancers based on analysis of The Cancer Genome Atlas data. Finally, knockdown of syntenin-1 inhibited the proliferation, migration and invasion of HNSC cells, and opposite findings were observed when syntenin-1 was over-expressed. Collectively, our studies indicate that syntenin-1 promotes invasion and progression of HNSC. It may serve as a valuable biomarker for lymph node metastasis or a potential target for therapeutic intervention in HNSC