The P13 Kinase Pathway In Cancer Biology Essay


Cancer is defined by an estimated 200 neoplasms marked with uncontrolled growth and spread of abnormal cells. Accounting for an estimated 7.4 million deaths in 2004 it is a group of diseases recognised for being one of the leading contributors to cancer death. Advances in therapeutic management of cancer have lead to effective developments in the control of primary tumours. By definition these are collections of cells harbouring genetic alterations at the site of origin. Metastasis is a principal event by which cells acquire a migratory phenotype allowing initial spread from this primary site to distant locations within the body. It is a process of lethality which primarily accounts for death of the cancer patients and yet has its molecular basis poorly understood [1].

The phosphatidylinositol 3-Kinase (PI3K) pathway is a conserved family of intracellular enzymes which phosphorylate the 3'-hydroxyl group of phosphatidylinositols and phosphoinositides. In eukaryotes this pathway regulates diverse collection of cellular processes including metabolism, survival, motility, proliferation, apoptosis, growth, migration and vesicle trafficking which in turn are involved in cancer. The signalling cascades initiated by these enzymes are often complex and a disruption in their regulation often results in generation of cancer.

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PI3Ks are defined on the basis of their substrate specifity and structure. Consequently, they are classified into 3 categorical groups denoted, class I, II, III. Class I PI3KS phosphorylate the inositol ring of phosphatidylinositol-3,4-biphosphate (PIP2) to phosphatidylinositol-3,4,5-triphosphate (PIP3) which is a potent second messenger responsible for intracellular signalling[4]. PI3Ks are further subdivided depending on signalling receptors which are used to activate them. Class 1A PI3Ks are activated by growth factor receptor tyrosine kinases (RTKs). Members of this class contain a P110 catalytic subunit (encompassing 3 alternating genes encoding 3 isoforms:,,) in association with a regulatory subunit (consisting of 3 isoforms, p85, p55 or p50). The Class 1B PI3Ks are activated by G-protein coupled receptors (GPCRs). The enzymes are found to be similar in structure and function to class 1A PI3Ks expressing, a p110-catalytic subunit but differ due to a p101 regulatory subunit instead of a p85 as seen in Class 1A[4].

Class II and III PI3Ks utilise phosphatidylinositol (PI) to synthesise PI-3-P. Clathrin is used as an adaptor protein in coated pits which further suggest a role in vesicular transport pathways. However till date no significant link has been established between class II PI3K pathway and its cellular function. One class III PI3K, VPS34, has been identified in mammals. Utilising available amino acids it entitles signalling to the Mammalian target of Rapamycin (mTOR) which in turn regulates growth of the organism and has a role in autophagy.

The class IA PI3Ks are found to harbour oncogenic mutations or gene amplifications and are specifically implicated in tumour progression . The further discussions will focus on this subfamily.

2.1 PI3-Kinase Activation

Class 1A PI3Ks are activated by the binding of ligands such as insulin; platelet derived growth factor (PDGF), heregulins (HRGs) and vascular endothelial growth factor (VEGF) to their respective tyrosine kinase receptors. In response to the extracellular cues, the p110 catalytic subunits are recruited to activated receptors via the regulatory subunits. Activated PI3Ks phosphorylate the inositol ring of PIP2 lipids to form the second messenger PIP3. In turn PIP3 lipids are responsible for regulating various downstream signalling processes. Alternatively, RAS can also bind to the p110 catalytic subunit to indirectly activate the PI3K [8].

2.2 PI3K Signalling

The lipid phosphatase PTEN (phosphatase and tensin homologue) is an important tumour suppressor protein . PTEN activity can be lost by mutations, deletions of methylation silencing at high frequencies in primary and metastatic human cancers. Its main substrate is PIP3, which it desphosphorylates into PIP2 and thereby, negatively regulates the PI3K by reducing downstream signalling. Otherwise, increasing levels of PIP3 recruit pleckstrin homology (PH) domain containing proteins to the membrane such as, AKT a serine/threonine kinase. Complete activation of AKT requires phosphorylation at two sites which are exposed during the interaction with PIP3. PDK1 (3-phosphoinositide-dependant kinase) phosphorylates at threonine 308 and PDK2 (complex rictor-mTOR) phosphorylates at serine 473. Activated AKT can phosphorylate a number of downstream targets that can result in cellular growth, survival and proliferation through various mechanisms and alongside other PI3K signalling pathways may be involved in PI3K-mediated tumourigenesis.

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Three members of serine/threonine AKT kinase family exist, AKT1, AKT2 and AKT3.Once activated AKT phosphorylate serine and threonine residues of targets, consequently, activating or inhibiting downstream signalling. As a result of PI3K activation these signalling cascades are linked to various downstream functions (figure 3).





2.3 PI3K pathway misregulation in cancer

PI3K activity in normal tissues is very tightly regulated. However, upregulation of the PI3K pathway is apparent in up to 50% of all human cancers. Aberrations in the PI3K signalling cascade include either overexpression or upregulation of PI3K or AKT isoforms or PTEN inactivation/silencing; all in turn leading to hyperactivation of the PI3K pathway . Much evidence has been collated in support for the link between PI3/AKT pathway in oncogenesis and tumour progression. Therefore, establishing the importance of pharmacological or molecular means of PI3K inhibition is necessary to prevent tumour cell proliferation and progression .

2.4 PI3K activation and cancer progression

Biological effects of PI3K over expression relating to cancer are divided into three categories:

1 - tumour growth (Cell proliferation, apoptosis, senescence)

2 - angiogenesis (production and response to angiogenic cytokines)

3 - metastasis (cytoskeletal plasticity, cell adhesion, cell motility, invasion)

2.4.1 - PI3K and tumour growth

PI3K can transmit many downstream signals via AKT which regulate the process of tumouriogenesis. However, cancer cells have developed anti-apoptotic mechanisms in opposition, to increase their survival chances. The ways by which AKT protects cells from death is likely to be multifactorial as it is involved in the phosphorylation of many downstream targets. AKT can phosphorylate MDM2, encouraging its translocation to the nucleus . This acts as a key regulator for the tumour surpressor p53 (which has a key role in carcinogenesis and cellular apoptosis) by promoting its proteasome mediated degradation. AKT can phosphorylate and inactivate the anti-apoptotic factor BAD as well as glycogen synthase kinase 3 (GSK3), which modulates modulates glucose metabolism and cell-cycle regulatory proteins. Alternatively, AKT blocks FOXO-mediated transcription of cell-cycle inhibitors and promotes the cell from G1 to S phase. It can also phosphorylate the transcription factor, 'nuclear factor-kappa B (NF-B) to enhance activity. Cell proliferation, size and growth are tightly regulated by the activation of the mammalian target of rapamycin (mTOR) via the PI3K/AKT pathway. TSC1 (tuberous scleoris complex -hamartin) and TSC2 (tuberin) form a complex to which results in inhibition of the G-protein Rheb. AKT can phosphorylate the TSC2 to relieve this inhibition imposed on Rheb activity. Consequently, this leads to the activation of the mTORC1. The TSC complex can be activated in response to low cellular energy, nutrients and stress. Energy deprivation states inactivate the action of mTORC1 via LKB1/STK11 (serine/threonine kinase 11) and AMPK (AMP-activated protein kinase). Restoration of cellular energy results through inhibiting the energy-consuming pathways such as mTORC1. In those cells lacking TSC1-TSC2 complex, nutrients and amino acids are capable of eliciting a response in mTORC1. Suggesting that nutrient input occurs further downstream of the TSC1-TSC2 complex . RAG GTPase are the nutrient sensing components of mTORC1. When excessive amino acids are present via the action of GTP loading, RAG activation occurs. RAG delivers mTORC1 to the pre-activated Rheb which in turn activates mTORC1.

2.4.2 - PI3K and angiogenesis

Angiogenesis is a process of new blood vessel development, from existing vasculature to meet the increasing demands of the growing tumour. This is an essential process for tumour growth beyond the size of 1-2mm in diameter. The process is tightly regulated via pro-angiogenic (e.g. Vascular endothelial growth factor and Fibroblast growth factor) and anti-angiogenic factors (e.g. Angiostatin and Endostatin), many which are produced by the tumour itself. The vascular endothelial growth factors (VEGF) are the most potent angiogenic cytokines which mediate tumour angiogenesis, in many cancers. The PI3K regulates the hypoxia-inducible factor 1 alpha (HIF-1) and VEGF. Hypoxic conditions induce the transcription of these factors and thereby increasing the oncogenic signals for tumour growth and angiogenesis . It is shown that additional loss of PTEN may upregulate this angiogenic pathway leading to increased VEGF levels, as a result of unregulated PI3K activity. Reintroducing PTEN or inhibiting PI3K using, 'LY294002' significantly have both shown to significantly reduce the VEGF factor production .

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VEGF and its two receptors, VEGFR-1 (Flt-1) and VEGFR 2 (KDR/Flk-1) which contain a cytoplasmic tyrosine kinase domain have been implicated in crucial regulation of blood vessel growth . It is shown that hypoxia leads to pronounced upregulation of these receptors . VEGF is secreted from tumour and host cells and targets the population of nearby endothelial cells, expressing high levels of VEGF receptors . PI3K is located downstream of receptors and upon activation of VEGF receptors, mediates a variety of function responses which collectively contribute to neoangiogenesis (figure x - adapted from ).

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Angiogenesis can cause cancer cells to acquire a more aggressive phenotype. Furthermore, it can facilitate tumour metastases by providing primary tumour cells with an efficient route of entry into the blood stream. Entry of cells is enhanced due to immature and highly permeable vessels which have little basement membrane and junctional complexes as compared to normal mature blood vessels .

3. PI3K and Metastasis - introduction

Metastasis is a complex process involving the spread of cancer cells to tissues and organs, distant from the primary site of tumour origination. When patients are diagnosed with cancer many of them present with detectable metastasis. A much greater proportion of patients will experience micrometases but currently, these are not clinically detectable . Cancer metastasis represents the major cause of morbidity and death for cancer patients. In fact, whereas primary tumours are often controlled and eradicated by the means of surgical and radiochemical treatments it is often the distribution of metastasised tumours throughout the body, which challenge therapeutic treatment . Metastasis is therefore characterised as the terminal step in malignancy and accounts for up to 90% of deaths in cancer patients.

It has long been accepted that malignant tumours show, organ-specifity. For instance, colon carcinomas tend to metastasise to the liver and lungs but rarely to the bone, skin, and kidneys whereas; breast carcinomas tend to metastasise to all these locations.

In 1889 an English scientist called Stephen Paget proposed a 'seed and soil' theory to explain the non random pattern of metastasis. His analysis documented that metastatic spread to distant locations within the body was not simply a random process but certain tumour cells (defined as the seeds) had a specific affinity for the microenvironment presented by other tissues within the body (defined as the soil) . His conclusion identified the need for the 'seed and soil' to be compatible in order for metastasis to occur. Therefore the ability of specific organs to provide these favourable environments and for specific tumour cells to respond to them dictated the metastatic development potential of different cancers .

In 1929 James Ewing opposed the 'seed and soil' theory and insisted that organ-specific metastatic dissemination occurs by mechanical factors which are a result from the anatomical structure of the vascular system located between the primary and secondary organs . Therefore, the organ with the highest vascular connections with the primary tumour would indeed as a result of tumour cell entrapment experience the highest metastatic colonisation. In contrast, organs with less vascular connections will experience a lower deposition of tumour cells and consequently develop less metastatic colonisation.

In 1970 detailed analysis of experimental mice studies defined that, the mechanical arrest of tumour cells could indeed occur in distance organs but the proliferation and growth of these secondary lesions were encouraged by specific organ cells .

The first stage of the metastatic cascade involves progressive growth of the primary tumour until a size of around 1-2 mm in diameter is reached. Neoplastic cell growth requires a constant supply of nutrients and these are provided by the surrounding extensive vascular networks. Angiogenic cytokines (e.g. VEGF) are secreted by tumour cells to encourage this capillary network development via the process of angiogenesis. The tumour cells eventually evade some of the host stroma. Thin walled venules such as the lymphatic channels offer little resistance to the tumour cells which the cells can effectively penetrate to enter circulation. Initially single cells enter the circulation with aggregates following and many undergo destruction throughout this process. Those cells which survive the circulatory destruction are trapped in capillary beds of distant organs. They attach themselves to either endothelial cells or subendothelial basement membranes which may be exposed. Extravasation occurs next, which involves entry of the cells into their destined organs. The cells begin to proliferate and this completes the metastatic process. To continue growth, as in the intravsation, development of another vascular network occurs. The cells can potentially re-enter circulation and travel to other distant locations, to form additional metastases (figure xx).



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3.1 - invasion

Epithelial derived tumours (carcinomas) may involve spatial or temporal occurrences of a process referred to EMT (epithelial mesenchymal transition) . EMT entitles polarised epithelial cells to escape from the rigid constraints presented by tissue architecture and adopt a mesenchymal phenotype. Essentially this includes down regulation of cell-cell adhesion, loss of polarity, enhanced migratory capacity, invasiveness, increased resistance to apoptosis and increased production of ECM components .

Invasion initiates the metastatic process and involves a change in the adherence of tumour cells to other cells and the extracellular matrix (ECM). The ECM forms a scaffold for cellular organisation. It has great control over cell behaviour and can dictate whether a cell will proliferate, migrate, remain stationary or undergo apoptosis . Before tumour cells can propel, proteolytic degradation and remodelling of the ECM occurs, with reduced cell-cell adhesion cellular progression throughout the tissue is facilitated . Cellular invasion therefore depends on the cooperation between adhesive and proteolytic mechanisms .

Tumour cell aherence to the ECM is mediated by integrins and by binding of these integrins to specific ECM proteins; signals can be transmitted into or out of the cell. Each integrin consists of two transmembrane subunits,  and . In mammals, 18 and 8 subunits associate in various combinations to form heterodimers that bind to ECM ligands . To date, atleast 20 integrins have been identified and their interactions with ECM ligands considered to be in an overlapping manner . The interactions recruit proteases belonging to two classes, secreted by either tumour or stromal cells. Metalloproteases (e.g. collagenases), are zinc dependant enzymes and serine-proteases (such as urokinase-type plasminogen activator) contain a highly reactive serine residue in their active site . The proteolytic enzymes are capable of degrading almost all the components of ECM whilst encouraging the pathway development for invasion and cancer cell migration over the matrix .

In addition to ECM degradation, tumour cells are understood to lower cell-cell adhesiveness to promote the metastatic potential of the tumour. Cell-cell adhesions are mediated by cadherin and desmosomal family members and these are essential for the cancer cells to remain in the tumour as a collective mass. Loss or dysfunction results in increased invasiveness and motility of the cancer cells. In particular, altered expression in 'E-cadherin' which promotes cell-cell adherence to 'N-cadherin' which is commonly expressed by mesenchymal cells to facilitate tumour cell binding to stroma during invasion has been identified .

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After successful ECM degradation and remodelling with lowered cell-cell adhesiveness tumour cells can undergo migration with more ease. Neoplastic cell entry into lymphatic and vascular channels for dissemination into circulation and metastatic growth in distant organs occurs . The migration mechanism utilised by the tumour cells is identical to that used by non-neoplastic cells in physiological processes such as embryonic morphogenesis, wound healing and immune cell trafficking .

For a cell to migrate modifications are required in the shape and stiffness in the cell body. This allows interaction to occur with the ECM which acts here, as both the substrate for migration and as a barrier towards the advancing cell body . The cell initially becomes polarised and elongates. A protrusion (e.g. lamellipoda, filopoda, pseudopods or invadopods) is adopted and the through extension of the cell's leading edge, attachment occurs with ECM components. Consequently, the leading edge attached to the ECM or the entire cell contract, generating a force large enough to create forward gliding motion of the cell body. The protusions which can be adopted by the cell body contain filamentous actin alongside structural and signalling proteins which entitle successful interactions with the ECM substrates. These extensions are necessary for cell motility and alongside natural occurrence they can be induced by chemokines and growth factors .

3.2 - angiogenesis

Tumour invasion is accompanied by angiogenesis. The vascular endothelial cell growth is often 20-2000 times faster in host-induced tumour epithelium than normal tissue . Besides the tumour expansion effect the most important way in which angiogenesis contributes to metastasis is by providing an efficient exit route for the tumour cells from the primary site into the blood circulation. Angiogenesis results in a high density of immature and highly permeable blood vessels with little basement membrane and fewer intercellular junctions, which further facilitates tumour cell penetration into circulation .

3.3 - intravasation

Tumour cells can often become detached from the primary site and their entry (intravasation) into lymphatic or vascular channels follow . The primary tumour may lack lymphatic supplies and therefore exit of tumour cells mainly occurs through intravasation into blood vessels . This entry into blood vessels requires direct penetration of the surrounding ECM and endothelial basement membrane. Adhesive interactions between the tumour cells and basement membrane are mediated by integrins and dissolution of this basement membrane follows resulting from proteolytic degradation. Newly formed blood vessels often have a defective architecture and their 'leakiness' tends to facilitate the tumour cell entry. Once the tumour cells pass the basement membrane they adhere to the vascular endothelial cells which retract and carry the tumour cells into circulation . The blood stream presents a harsh environment for metastasising tumour cells due to, velocity-induced shear forces, lack of a substratum and the presence of immune cells. The rate of shedding can be as large as 1 million cells per day for rapidly growing tumours that are at least 1cm in size . Tumour cells are discharged into the vascular system in the form of single cells and clumps. Only a small percentage (<0.01%) of these cells survive to initiate metastatic colonies and these are often shielded cells located towards the centre of the clumped aggregates .

3.4 - arrest

Circulating cells eventually arrest in the target organ and this is a pre-requisite for their entry into extravascular space . The cells use a variety of mechanism to arrest in the vessels of the target organ including mechanical wedging, entrapment with platlets and fibrin, and attachment to target organ endothelium via specific tumour cell surface receptors .

3.5 - extravasation

The tumour cells can adhere to the endothelial lining. The endothelial cells being to retract and in combination with proteolytic damage of the basement membrane, tumour cells can establish a secondary tumour at the target site. Evidence suggests that tumours can proliferate intravascularly without the need to extravasate .

3.6 - secondary tumour growth

The extravasated cells proliferate as colonies but require an expansion in blood supply to meet their growth requirements. Angiogenesis is therefore an essential requirement at the end of the metastatic cascade as well as the beginning. Host and tumour factors can alter the microenvironment required for growth and survival. Tumour cells can synthesise and secrete their own growth factors to encourage their development into a secondary tumour .

4 - Hypoxia - introduction

Tissue hypoxia results from an inadequate supply of oxygen which compromises biological functions. Hypoxia is considered of having a fundamental role in solid tumours and is primarily a consequence of structurally and functionally disturbed microcirculation and the deterioration of diffusion conditions. Tumour hypoxia appears to be associated with propagation, malignant progression, resistance to therapy and therefore it has become a central issue in tumour physiology and cancer treatment .

Tumour hypoxia is considered to be a therapeutic problem because tumour cells gain resistance to ionising radiation. Additionally, unperfused regions also contribute towards the resistance of some chemotherapeutic agents .

Sustained hypoxia leads to the occurrence of cellular changes which can develop characteristics of a more aggressive phenotype . Tumour hypoxia is associated with a poor prognosis, enhanced metastatic potential, greater malignant progression and increased local invasiveness . Likewise, an increased resistant to radiation and other cancer treatments also occur .

4.1 - causes of hypoxia

Tumour hypoxia results from an imbalance between the cellular oxygen consumption and oxygen supply to the cells. Development of tumour hypoxia can be perfusion, diffusion or anaemia related .

Perfusion related (acute) hypoxia occurs due to an inadequate distribution of oxygen in the tissues. Tumour microvasculatures frequently experience structural and functional abnormalities, such as disorganised vascular networks, dilated vessels, an elongated and tortuous shape, an incomplete endothelial lining, a lack of physiological and pharmacological receptors, an absence of flow regulation and intermittent stasis . tumour . In turn, these components cause irregular blood flow, periods of haemostasis and a perfusion limited lack of oxygen delivery results in ischaemic hypoxia, which is often transient .

Diffusion related (chronic) hypoxia occurs due to increased diffusion distances due to tumour expansion. The increasing distance between tumour and the nutritive vessel causes for the interstitial pressure of oxygen to decrease. When the distance exceeds 70m there tends to be an inadequate oxygen supply whereas at a distance of around 150-180 m the cells become anoxic. Thus, enlarged distances in tumours consequently generate regions of hypoxia which are heterogeneously distributed throughout the tumour .

Anaemic hypoxia is based on the reduced oxygen carrying capacity of the blood. It is shown that even moderately reduced haemoglobin levels of 10 to 12 g/dl can significantly reduce tumour oxygenation. Anaemic hypoxia is intensified especially when the low oxygen transport capacity coincides with a low tissue perfusion rate. Similar effects are seen in heavy smokers due to the formation of carboxyhaemoglobin as carbon-monoxide blocks the haemoglobin oxygen carrying capacity .

4.2 Hypoxia and PI3K pathway

Tumour cells are often in an oxygen deprived environment and undergo various genetic and adaptive changes allowing survival, proliferation and disease progression. The molecular mechanisms underlying these adaptive changes involve in the transcription of a number of genes which either, increase the availability of oxygen to the tissues or reduce cellular consumption of oxygen through activation of glycolysis via the glucose transporter 1 (GLUT1).

Hypoxia inducible factor-1 (HIF-1) is one of the most important hypoxia activated factors which regulates genes involved in cancer progression, metabolic adaptation, angiogenesis, metastasis and resistance to apoptosis . It is composed of two subunits, the oxygen sensitive HIF-1 and the constitutively expressed HIF-1. Under normoxic conditions HIF-1 is inactivated by hydroxylation. This enables molecular recognition and binding with the Von Hippel-Lindau protein and the complex is degraded by the 26S proteasome mediated ubiquitination . It is apparent that Hsp90 protects HIF-1 from oxygen-independent degradation. Studies implicate that the PI3K pathway is required HIF-1 stabilisation via heat shock proteins . Following translocation into the nucleus and heterodimerisation with HIF-1 and additional co-factors, activation of the HIF-responsive genes occurs .

PI3K pathway which is one of the well characterised cell survival signalling pathways has been implicated in the control of the HIF-1 protein expression . Loss of PTEN or deregulation of growth factor signalling increased PI3K activity. A number of studies suggest that activation of the PI3K pathway exerts increased HIF-1 translation through an mTOR independent pathway . Therefore, the PI3K pathway is considered to be involved in tumour cell acquired resistance to apoptosis in hypoxia .

5 - Thyroid cancer - introduction

Thyroid carcinomas represent around 1% of newly diagnosed cancers and although uncommon they represent most common type of endocrine malignancy. Although, benign disease of the thyroid is relatively common, malignant growth is fairly rare. The incidence rates vary from 0.5 to 10 cases per every 100 000 individuals in the population .

Carcinomas derived from follicular epithelial cells can be categorised into well-differentiated, poorly differentiated and undifferentiated carcinomas .

Well differentiated carcinomas, consist of papillary (PTC) and follicular thyroid carcinomas (FTC) , which account for more than 90% of thyroid malignancies . They are usually treatable and have a high cure rate.

PTC tends to be the most common type of thyroid malignancy and with an overall survival of above 90% is associated with an excellent prognosis . PTC spread through the lymphatics within the thyroid to regional lymph nodes in the neck and very rarely spread haematogenously to the lungs, liver, bone and brain .

FTC accounts for 10-32% of differentiated thyroid carcinomas. Similar to PTC, follicular carcinomas are common in elder patients and usually associated with advanced disease and a poorer survival rate . FTC have a lower lymph node involvement and tend to metastasise to the lungs and bone via haematologic spread .

Anaplastic thyroid carcinomas account for around 1-2% of all thyroid cancers . Although less common, they tend to be aggressive, metastasise early, have a poorer prognosis and account for 14 - 39% of all thyroid carcinoma deaths . They are undifferentiated tumours which more commonly manifest in elderly females as an enlarging mass in the neck . Although fairly uncommon, the aggressive metastasising nature of anaplastic tumours makes them extremely fatal. Consequently they are unresectable and many powerful systemic therapies have proven ineffective with the mean survival time being often less than six months .

5.2 PI3K and thyroid cancer

Research over many years has implicated an important role for the PI3K-AKT signalling pathway in the development and progression of a range of tumours including, thyroid cancer. Although multiple mechanisms exist by which aberrant activation of PI3K signalling is initiated, the resulting downstream signalling events which occur, are similar.

Much supporting evidence exists implicating the role of mutated PI3K pathway proteins in the development of thyroid cancer including the PI3 Kinase itself. For instance, Cowden disease, an autosomal cancer predisposition syndrome is associated with and elevated risk of developing thyroid neoplasia. The disorder is initiated by the inactivating mutations of the PTEN tumour suppressor gene. The high frequency of mutations and gene transformations in upstream signalling molecules, such as, RAS, RET and PTC reinforce the PI3K pathway involvement in the development of malignancies as do the more recently identified mutations of the PI3KCA and AKT1 .

5.3 Therapeutic targeting of the PI3K pathway

As PI3K overactivation is commonly implicated in many cancers including thyroid, intense efforts have been made to inhibit certain proteins in the cascade. The development of selective inhibitors of the PI3K signalling is currently and intense area of research. Although the pathway presents many targets for intervention, complexities in signal regulation is a substantial challenge in therapeutic design.

In relation to the therapeutic management of thyroid cancer, Rapamycin has been shown to inhibit PTEN loss-related thyroid cell growth in both vivo and vitro. Whereas more specific targeting of the PI3K itself using inhibitor compounds such as Wortmanin (selective) and LY294002 (non-selective) has also been carried out in vitro.

GDC 0941 is more recent development in the newer generation of the PI3K inhibitors. It is a selective and very potent inhibitor of class 1 PI3Ks and has shown significant in vivo antitumor activity. The role of aberrant PI3K signalling as a mediator of thyroid cancer development and progression requires further experimental confirmation. This novel study is aimed to determine the effect of the PI3K inhibitor GDC0941 on cell migration, a process known to be regulated by the PI3K pathway, under varying oxygen tensions.

Materials and Methods

6.0 introduction

The scratch wound migration assay will be utilised to analyse the migration of the thyroid cancer cell lines in vitro. Prior to experimental working all worktop surfaces, aspirator tubes, glass pipettes, tweezers, glass cover-slips and gloves were sterilised using 70% ethanol. The tweezers were used to place sterilised cover-slips into the petri dishes.

6.1 materials

70% ethanol, 20mm * 20mm glass cover slips, T75 flask, 35mm petri dishes, 5ml/10ml/20ml pipettes, trypsin solution, virkon solution, phosphate buffered saline (PBS), dimethyl sulfoxide (DMSO), haemocytometer, RPMI 1640 medium, foetal calf serum, L-glutamine, 10% formalin solution, DAKO fluorescent mounting media, nail varnish, 8505C and FTC133 cell lines.

6.2 culturing of cells

The two cell lines that were cultured were 8505C and FTC133. The cell lines were grown under similar conditions of 37°C, 5% CO2 and 95% humidity in T75 flasks with complete medium. The complete medium consisted of, 85% RPMI-1640 essential medium, 10% foetal calf serum and 5% L-glutamine and approximately 12ml of the mixture was used to culture the cells. This complete culture medium provided the nutrients required for optimal growth and to satisfy the growth requirements of the cultured cells such as, L-glutamine, amino acids and growth promoting factors. The T75 flasks used provided an area of 75cm2 were used to culture the cells until at least 90% confluent cell growth was observed.

6.3 passaging of cells

Trypsin is a common protease that is utilised to degrade the anchoring proteins which bond the cells to the flask. However, the calf serum contained within the medium has antitrypsin activity making it is essential to initially aspirate this medium from the flask. Two washes with Phosphate Buffer Solution (PBS) ensure the complete removal of any remaining calf serum that would confer antitrypsin activity. During each wash, 5ml PBS was inserted into the flask which was tilted to ensure coverage of the cell binding surface and then removed. 1.5ml of Trypsin solution was then added to cleave cells from the binding surface. The flask containing the Trypsin solution was placed in the incubator at 37°C for 3 to 4 minutes, to optimise enzymatic trypsinisation and after removal from the incubator the flask was tapped to enhance cell detachment. Cell separation and detachment was confirmed by visual analysis and by using light microscopy. Using a 5ml sterilised graduated pipette, 4ml of the complete culture medium (85% RPMI-1640, 10% foetal calf serum, 5% L-glutamine) was added to the flask to neutralise the trypsin activity. The resulting cell suspension (minus 1ml of the suspension left in the flask) was removed using a pipette and inserted into a 10ml capped tube. To facilitate mixing of the cell suspension it was drawn up and down using a pipette several times. Consequently, this encouraged the breakdown of any cellular clumps creating a single cell suspension.

6.4 counting of cells

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