Nanomedicines and Ovarian Cancer 

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High Grade Serous Ovarian Cancer (HGSOC)

Risk Factors

Ovarian cancer most often affects women between the ages of 75 and 80 years 10,11. Two hypotheses have been suggested to explain the development of High Grade Serous Ovarian Cancer (HGSOC). Firstly ovulation creates a lesion of the ovarian epithelium that needs to be repaired. This highly inflammatory microenvironment may lead to DNA damage, replication errors and malignant transformations12,13. Incessant ovulation with an early menarche has been related to an increase prevalence of low grade ovarian cancer14,15.
Secondly, the development of Ovarian Cancer has been related to the onset of menopause. During menopause the ovaries are unable to respond to hormonal stimuli stopping the feedback of gonadotropins16. The higher level of oestrogen compared to progesterone then results in higher oestrogen exposure by ovarian epithelial cells increasing the risk to develop ovarian cancer5,17. Obesity is another risk factor due to high androgens which can be converted into oestrogen in adipose tissue by aromatase11,17,18. A decrease in blood sex hormone binding globulins can also result in an increase in the relative amount of free oestrogen. Finally, the use of hormonal therapy such as infertility drugs (gonadotropin releasing-hormone antagonists or clomiphene)16,17 have been determined as risk factors.
Hereditary factors are also linked to an overall 5% to 10% 17 risk of developing OC when one immediate relative has had OC . This is due to the inherited mutation of BRCA genes which increases the chance to develop OC for women of the age of 70 with BRCA1 mutation by 63% and by 27% for BRCA2 mutation19. BRCA1 and 2 are involved in DNA repair, maintenance of genome stability and function as tumour suppressors20. The use of progesterone-based oral contraceptives for at least 4 years has been related to a decrease of ovarian cancer risk by 50% in women with a BRCA mutation21 by blocking ovulation. Genetic mutations are not limited to BRCA1/2 genes inducing instability in genome22 with deficiencies in homologous recombination, impairing the repair of the DNA also reported23. Several suppressor genes and oncogenes have been associated with ovarian cancer. P53 and mismatch repair (MMR) or double strand break repair system (CHEK, RAD1) mutations have also been related to cancer development4,11,17.
Finally, epigenetic modifications have also be related to malignant development and progression of ovarian cancer. Hypermethylation of BRCA1 and 2 promoters has been related to a decrease in the efficacy of DNA repair of spontaneous mutations in ovarian epithelial cells24. Hypermethylation of CpG islands have been found to be related with tumour development in comparison with normal tissues25.

Metastasis

Cancer metastasis is the leading cause of cancer death accounting for 90% of ovarian cancer cases26. Ovarian cancer has a unique mode of development, disseminating locally in the peritoneal cavity and rarely beyond27,28. Peritoneal dissemination occurs by movement of ovarian cancer cells in the peritoneal fluid (also called ascites)29. Dissemination happens either when the tumour has grown extensively in the organ and caused rupture of the ovary surface or when tumour arises from the surface of the ovary. This dissemination is accompanied by molecular alterations in cells and notably through a cadherin switch involving overexpression of E-cadherin (Figure 2), and activation of N-cadherin expression which is a mesenchymal marker and vimentin expression30. Moreover the phenotype of the cells is modified as they undergo an epithelial to mesenchymal transition 26,31.
Once in the peritoneal cavity, ovarian cancer cells undergo two different paths. Isolated cells undergo anoikis as they lost interaction with extracellular matrix and other cells while multicellular aggregates32 formed in the peritoneal cavity form spheroids that can seed in multiple distal sites. The invasion of secondary sites is facilitated by the remodelling of the extracellular matrix of the mesothelial lining at these locations by matrix metalloproteases.
Dissemination of ovarian cancer cells from the primary tumour to the peritoneal cavity through ascitic fluid is accompanied with cadherin switch allowing the formation of spheroids which can land on the mesothelial lining of the abdominal cavity. This attachment leads to a second set of modifications of the cellular properties in their interactions with other cells and cancer cells acquire the ability to go through the peritoneum.
The colonisation of secondary sites involves the interaction between ovarian cancer cells and mesothelium cells of the peritoneal cavity28 switching the cancer cells from a proliferative to an invasive phenotype that is translated by an increase of integrin expression33.
The adhesion of the spheroids on the surface of the mesothelium causes a decrease of E-cadherin and increase of CD4434,35. This docking triggers the expression of fibronectin by the mesothelium increasing the interaction with the integrin of the cancer cells36. CD44 and L1CAM are crucial for secondary tumour formation35. Blocking CD44 or L1CAM expression has been shown to reduce mesothelial adhesion37 (Figure 2). Once docked the spheroids initiate infiltration and spread to surrounding tissues.
The dissemination through the peritoneum is a passive mechanism involving the circulation and accumulation of ascitic fluid27. In comparison with the surrounding environment of other solid tumours, the malignant ascitic fluid accumulating in the peritoneal cavity during ovarian cancer progression is uniquely constituted forming of highly inflammatory environment due to macrophage activation38. The circulation of ascitic fluid transports the spheroids allowing them to spread and attach throughout the peritoneal cavity forming nodules mainly on the omentum but also on the diaphragm, liver or lungs23,39. The ascitic fluid is constantly changing with the evolution of the pathology and plays a major role in tumour progression, spheroid formation, tumour dissemination. For example, lysophosphatic acid which is present in ascites or ovarian cancer patients promotes motility and invasiveness of cancer cells via induction of expression of metalloproteases that modify the extracellular matrix of the mesothelium33. Moreover the increase of CXCL12 released by epithelial ovarian tumoral cells in the ascitic fluid acts as autocrine and paracrine stimulation inducing increased expression of integrins by ovarian cancer cells leading to increased migratory potential. Finally after cancer cell implantation, synthesis of pro-inflammatory TNF-α by ovarian cancer cells stimulates endothelial cells40 to secrete interleukins enhancing angiogenesis at metastatic tumour sites. The accumulation of ascitic fluid is not well understood but it is thought that vascular endothelial growth factor (VEGF) is involved41. VEGF promotes angiogenesis inducing in vitro the formation of confluent microvascular endothelial cells that invade collagen gels and form capillary structures. Its overexpression has been detected in some cancer patients and allow the creation of a more favourable environment for the new implant.

Different forms of Ovarian Cancer

Ovarian cancer cells can originated from cells of ovarian epithelial surfaces42, from the epithelium of distal fallopian tubes 43 or from peritoneal cavity epithelium26. Epithelial ovarian cancer is the most common tumour type accounting for 90% of the cases44. They are separated in 2 categories depending on the pathway of tumorigenesis (Histotypes are detailed in figure 3).
Type I is comprised of low grade serous, endometrioid, mucinous and clear cell carcinomas originating from lesions in the ovary45. They are slow growing tumours and characterized by a stable genome and do not carry a p53 mutation, however they have a mutation in KRAS gene, which is involved in the RAS/MAPK pathway controlling cell growth, proliferation or maturation45. Type II are high grade serous or undifferentiated carcinomas and carcinosarcomas. They are fast growing tumours with a high metastatic potential and a low level of detection. They represent about 75% of the Epithelial Ovarian Cancer diagnosis46,47.
Recent studies have been suggesting that High Grade Serous Ovarian Cancer originate from the distal end of fallopian tubes before crossing the surface epithelium of the ovary43. They are characterized by the absence of architecture and dysmorphic nuclei48. Other features include high nuclear to cytoplasmic ratio, atypic mitotic figures49, high mitotic/apoptotic rates50 and a high Ki-67 protein concentration51.
Ovarian cancer is defined by any any primary malignant tumour initiating from the ovary, the endometrium or from fallopian tubes. More than 85% of ovarian cancers are carcinomas, meaning they are derived from epithelium. Amongst them 70-74% are High Grade Serous Carcinoma, 3-5% are Low-Grade Serous Carcinoma, 10 to 26% are Clear Cell Carcinoma, 2-6% are Mucinous Carcinoma and 7-24% are Endometrioid Carcinoma.

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Biomechanical process during cancer progression

Dissemination of cancer cells following Epithelial to Mesenchymal Transition (EMT) is sustained by modification of cell-cell, cell-matrix interactions and cytoskeleton modifications. The cell cytoskeleton is formed of actin filaments, microtubules and intermediate filaments that all influence cell morphology53. These different structures interact with each other providing mechanical stability of cells. The effect of chemical drugs targeting cytoskeleton been used to reveal the role of each type of fibre in cell elasticity. For example the depolymerization of actin fibres resulted in rounder cells related to softening of the cells54.
Invasive properties in HEY/HEYA8 ovarian cells have revealed that modified morphology is linked to cytoskeleton modifications increased the migration capacity of these cells55–57. Such observations are not directly applicable to in vivo mechanisms as in the tumour environment cell-cell contacts and extracellular matrix are strongly affecting cell stiffness58–61. Notably during the metastatic process, the cells acquire motility and increased deformability. Those morphological changes influence cell stiffness55.
The adhesion of cells to the extra cellular matrix is a key property that has important functions in cell physiology. Indentation experiments in breast epithelial cancer cells have shown that matrix stiffness dictates intracellular mechanical state of those cells62. Moreover the comparison between cirrhosis tissue and hepatocellular carcinoma tissue did not show any stiffness differences suggesting the hardness of the liver is increased during carcinogenesis63.
Such modifications are studied using instruments, such as the atomic force microscope (AFM) which can measure nanomechanical changes. The measure of a force necessary to indent a cell (Young’s modulus) in different cancerous cells such as breast64,65, prostate64,66, ovaries67, or kidney58 cancer has been shown to be less than their normal counterparts. It appears that modification of the Young’s modulus can indicate the transition to a cancerous state for individual cells.

Current diagnostics and new developments

Current diagnostics

Different symptoms of early stage ovarian cancer have been identified, however, they are not specific to ovarian cancer, including abdominal and pelvic pain, irregular menarche, change in bowels habits and increased urinary frequency5. These benign gastrointestinal and gynaecological problems are often symptoms attributed to stomach or colon diseases.
The familial history of cancers plays an important role in deciphering the cancer risk. Importantly it can be related to the presence of an inherited mutation in the germline such as a BRCA genes mutation68.
Ovarian cancer detection is currently based on circulating cancer antigen 125 (CA-125) glycoprotein concentrations as is has been shown to be elevated in 50% of cases with early stage ovarian cancer69, but is also increased in pregnancy and endometriosis and other benign clinical conditions70, which reduces its specificity. The lack of specificity and sensitivity of current early detection biomarkers severely impacts screening efficacy71,72. Population screening is also limited due to the rarity of the disease, and therefore cost implications related to such testing. However CA-125 remains an effective approach for sequentially monitoring the response to chemotherapy from patients and detecting relapse208.
Transvaginal ultrasonography (TVU) is used in addition with CA-125 to screen symptomatic patients to detect ovarian cancer and rule out the false positives caused by weak or absent CA-125 signal. TVU enables precise imaging of the ovaries and helps to identify simple cysts, complex pelvic masses and solid tumours. However, only a fraction of metastatic tumours reach a sonographically-detectable size which may lead to false negatives in the detection of early-stage ovarian cancers74.
Additionally, magnetic resonance tomography imaging (MRI) can be used when the other two tests give opposing results. The low spatial resolution of ovarian cancer hinders the detection of small tumours75. If the result of the diagnostic test raises suspicions, surgical approaches are adopted depending on the stage of the tumours76.
The staging system (Table 1) for ovarian cancer is derived from the International Federation of Gynaecology and Obstetrics77.

Development of a new blood-based biomarker

Stable and radioactive isotopes have been used in earth science in numerous fields (paleoclimate, paleocirculation, chemical evolution of earth, pollution). Recent improvements of Multi-Collector Inductively Coupled Plasma Mass Spectrometry (MC-ICP-MS), thermic ionization (TIMS) and isotopic ratio mass spectrometry (IRMS) allowed new measurements of small isotopic elemental variations. While those techniques were common in archaeology, they have recently been used in human and animal medicine78–81. Alkaline earth metals such as Calcium and Magnesium and the transition elements Iron, Copper, and Zinc have been studied due to their functional roles in biology and because their turnover rates in the body are relatively short.
Dietary intake of copper in human needs to be around between 1 and 3mg/day82,83. A portion is absorbed by the intestinal cells, after being reduced from Cu2+ to Cu+ by the membrane protein STEAP84, through the CTR1 transporter. In cells, chaperons ATOX1 or COX17 85,86 bind to copper Cu+ and deliver copper to different organites where it is used as cofactor of cytochrome c oxidase in mitochondria, or the superoxide dismutase SOD1 that catalyses the scavenging of ROS producing oxygen and hydroperoxide. Copper is transported through the intestinal cells and delivered to the blood through the ATP7A copper transporter85. Copper is then transported in blood by the ceruloplasmin to the liver87. The liver is the main site of copper accumulation, controlling concentrations in blood88. Liver synthesizes ceruloplasmin89 which can transport up to seven copper90 atoms due to a methionine rich domain and cysteine-histidine domains. Excess copper is excreted in the duodenum or in urine via the kidneys. Demands of copper in organs depend on their metabolic functions such as mitochondrial content and activity. For example in muscles, the high amount of mitochondria increase the demand of copper for cytochrome c function. This transmembrane protein contains 2 copper centres91. Their functions are to transport electrons from the soluble cytochrome c to the oxygen that is reduced into water.
Modifications of Cu concentration and relative abundance of Cu isotopes (fractionation) have been linked to modified metabolic processes (oxidative phosphorylation, hypoxia) or in angiogenesis, and thus to health and disease92. In different cancers it has been shown that copper is required for angiogenic processes93, stimulating proliferation and migration of endothelial cells94. In the liver tissue of colon tumour bearing mice, gene expression the copper transporters ceruloplasmin, and CTR1 and ATP7B was increased significantly, which can explain elevated copper serum levels95 and suggesting its potential use as a diagnostic marker of cancer. Isotopic ratio between heavy isotope 65Cu and the light isotope 63Cu has been measured in different healthy and human materials96 (Table 2).

Table of contents :

INTRODUCTION 
I. Ovarian cancer
1. The disease
2. Biomechanical process during cancer progression
3. Current diagnostics and new developments
4. ‘Classic’ Ovarian Cancer treatments
5. ‘New’ Ovarian Cancer treatments
6. Nanomedicines and Ovarian Cancer
II. Epigenetics
1. Definition
2. Transcription activation mark (H3K4)
3. Transcription repression mark (H3K27)
4. Heterochromatin mark (H3K9)
5. Current knowledge of the effect of epigenetics in ovarian cancer
III. Selenium in cancer treatment
1. Dietary window of selenium and metabolism
2. Low dose of selenium triggers ROS scavenging
3. High doses of selenium are cytotoxic
4. Effect of selenium on epigenetic mechanisms
5. Selenium Nanoparticles
IV. Objectives
SELENIUM NANOPARTICLES TRIGGERS ALTERATIONS IN OVARIAN CANCER CELLS BIOMECHANICS 
I. Presentation of the article
II. Article
SELENIUM NANOPARTICLES INDUCE GLOBAL HISTONE METHYLATION CHANGES IN OVARIAN CANCER CELLS 
I. Presentation of the article
II. Article
CU ISOTOPE RATIOS ARE MEANINGFUL IN OVARIAN CANCER DIAGNOSIS 
I. Presentation of the article
II. Article
CONCLUSION AND PERSPECTIVES 
I. Conclusions
II. Perspectives
REFERENCES 

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