Abstract
Ovarian cancer (OC) is a major concern among the gynaecological cancers due to its high mortality rate. Women with OC rarely present with specific symptoms, and accurate strategies for the screening of OC with high sensitivity and specificity involving biomolecular markers and advanced imaging techniques are still under development. Thus, OC is mostly diagnosed at the late stage of the disease when the clinical outcomes are poor. The standard management of patients with OC is debulking surgery and a combination of chemotherapeutic regimens. The initial response to these treatments is high, but recurrent disease usually occurs. When patients relapse, they may not respond to the same regimen of chemotherapy drugs. Therefore, other strategies with combined treatments targeting active signalling pathways in OC are strongly justified, including targeting pathways that promote tumour cell proliferation, invasion, metastasis, and the development of chemoresistance.
Interestingly, the majority of women with advanced stage OC develop ascitic fluid in their peritoneal cavity, and this is associated with poor prognosis. The cellular and acellular components of this malignant ascites exhibit a proinflammatory milieu which promotes tumour cells proliferation, invasion, and metastasis, as well as development of chemoresistance. Abundant cytokines and growth factors, such as interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-α), or epidermal growth factor (EGF) in ascitic fluid have been associated with the activation of major survival signalling pathways in OC, including the janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Evidence that supports targeting the JAK/STAT pathway comes from immunohistochemical evaluation which demonstrates constitutive activation of STAT3 in the tissue of patients who develop chemoresistance. Thus, targeting the JAK/STAT pathway is a plausible option to reduce OC cell viability.
The overall aim of this thesis was to investigate the effects of JAK1/2 inhibitor ruxolitinib in OC cell lines (SKOV3, ID8 and OV90) in the absence or presence of ascitic fluid. Furthermore, this study aimed to determine the mechanism of action of ruxolitinib, investigate other activated signalling pathways that may result in resistance to ruxolitinib, as well as identify an agent to use in combination to augment the effect of ruxolitinib.
The JAK/STAT pathway is well known for responding to cytokines and chemokines in an inflammatory niche. The ascitic fluid found in OC patients is akin to an inflammatory condition. In this study, the hypothesis was that ruxolitinib would effectively reduce OC cell viability. A further hypothesis was that ascitic fluid would attenuate the efficacy of ruxolitinib via activation of compensatory signalling pathways, and in order to provide a clinically useful response, ruxolitinib may be best utilised in combination with another drug. The three-dimensional (3D) model is more physiologically relevant to the tumour microenvironment (TME) and therefore using 3D culture models are considered critical in this study.
The findings in this study have demonstrated that ruxolitinib significantly reduced OC cell viability in a monolayer model. However, in a 3D cluster model, ruxolitinib showed limited activity. The presence of ascitic fluid attenuated the effect of ruxolitinib in both culture models. Furthermore, based on results of a phospho-kinase proteomic array and pathway enrichment analysis using the OV90 cell line (which represents a high-grade serous OC subtype), the PI3K/Akt/mTOR signalling pathway has been identified as one of the major activated compensatory survival pathways following ruxolitinib and ascitic fluid treatment. These findings justified the use of the mTOR inhibitor everolimus in combination therapy with ruxolitinib. This study demonstrated that combination therapy of ruxolitinib with everolimus significantly reduced OV90 cell viability when compared with ruxolitinib monotherapy, despite a lack of evidence for synergistic or additive effects. However, combined treatment using both drugs was no more effective than everolimus treatment alone. In addition, the presence of ascitic fluid did not reduce the efficacy of the combination therapy.
This study has demonstrated the importance of culture microenvironment in the exploration of potential cancer drugs for use in OC. However, there was no evidence that combination therapy of ruxolitinib and everolimus provided therapeutic benefit over everolimus monotherapy. Hence, further work is required to evaluate this combined therapy on different cell lines or models and explore the potential of other combination drugs targeting JAK/STAT and PI3K/Akt/mTOR signalling.