The interaction of wnt-11 and signalling cascades in prostate cancer

A thesis submitted to the University of Bedfordshire, in fulfilment of the requirements for the degree of Masters by ResearchCastration resistant prostate cancer proposes an array of issues in terms of treatment options. It is therefore necessary to decipher the underlying mechanism involved in androgen independent prostate cancer and neuroendocrine differentiation, which is associated with malignant and metastatic disease. Wnt-11 has been previously shown to be associated with the more malignant version of the disease by being involved in the cellular proliferation and differentiation of the cancerous cells. The methodology adopted to identify signalling pathways triggered upon Wnt-11 activation were inhibition of the JNK, PKA, PI3K and mTOR pathway with various concentrations of inhibitors, proceeding this proliferation, migration and gene expression experiments were carried out three times, each experiment containing a triplet of each condition. Results collected were significant in all experiments excluding the proliferation results involving the PKA pathway. A preliminary mechanism was established between Wnt-11 and the mentioned pathways with neuroendocrine differentiation. The experiments carried out along with the correlated data were novel and brings research one step closer to understanding the mechanism of androgen independent prostate cancer, which in turn can hopefully relay to new therapy options which are currently absent on the market

Mechanisms involved in the development of androgen independent prostate: Androgen receptor activation leads to a dissociation of E2F1:PHB in prostate cancer

Prostate cancer (PC) is the most common cancer found in men in the Western world. Predominantly, the pathogenesis of PC is driven by aberrant androgen receptor (AR) signalling, resulting in heightened cell proliferation and cell survival. Current treatment options target the AR signalling cascade by androgen ablation therapy, whereby anti-androgens antagonistically bind to the AR. This prevents AR activity, thus inhibiting transcription of genes involved in cell proliferation, survival and metastasis. However, eventually androgen ablation therapy leads to relapse, as the cancer succumbs to a low androgen environment, leading to a poorer prognosis with limited treatment options, otherwise known as ‘castration resistant PC’ (CRPC). This is where new clinical interventions are urgently required. One mechanism that could allude to androgen independent PC, is the down-regulation of AR co-repressors. One such co-repressor is known as Prohibitin (PHB). PHB is ubiquitously expressed in mammalian cells and displays anti-proliferative functions. Prior to this project, it was identified that AR signalling leads to the down-regulation of PHB at both a transcript and protein level and causes PHB to dissociate from the chromatin. However, over-expression of PHB leads to the down-regulation of androgen responsive genes, and its increased association with the chromatin. Thus within PC, both the AR and PHB are in a dynamic opposition. This project identified that PHB has a repressive role on the cell cycle by holding the cell population within the G1 phase. RNA-sequencing and Q-PCR data identified that PHB had a significant repressive role on gene families involved in DNA replication and cell cycle regulation and progression. Such gene families include cyclins, E2Fs and MCMs. MCMs are gene targets of E2Fs, and therefore it was assessed if PHB had an effect on the promoter activity of two MCM genes; MCM5&6. Interestingly, with PHB over-expression, repression of both MCM5&6 promoter activity was seen. It was then identified that PHB physically interacts with E2F1 in androgen responsive PC cells. However this interaction was reduced upon androgen stimulation of the AR. This led to the preliminary mechanism that PHB can bind to E2F1, potentially preventing E2F from binding to MCM promoter regions, leading to an inhibition of the cell cycle in PC cells. Moreover, it was identified that androgen stimulation led to a likely dephosphorylation event happening to the PHB protein that could explain how PHB dissociates from the chromatin and E2F1. Signalling pathways that could lead to the non-genomic activation of the AR was narrowed down to the Src-pathway highlighted by a Kinexus™ protein array. Interestingly, inhibiting the Src-pathway stops PHB’s likely dephosphorylation event. Accumulating previous data and data collected throughout this thesis, PHB up-regulation leads to cell cycle arrest through a direct interact of a key cell cycle regulator; E2F1. Moreover, PHB was found to have an inhibitory effect on both the migration and adhesion of PC cells, potentially through the down-regulation of wnt genes. Thus potentially, PHB up-regulation can inhibit the migratory potential of PC cells, stopping the progression of the disease

Unveiling the potential of prohibitin in cancer

Recently, research has shed new light on the role of Prohibitin (PHB) in cancer pathogenesis across an array of cancer types. Important mechanisms for PHB have been unveiled in several cancers, especially with regard to the androgen independent state of prostate cancer (PC) and oestrogen dependent breast cancer. However, PHB is often overlooked due to its complex but subtle roles within the cell. Having gathered both historical and current research exploring PHB's role in different cancer types including prostate and breast, here we aim to pair this information with its molecular properties in the hope of translating this information into a clinical perspective, thus discussing its possible use in future cancer therapy

Targeting Wnt signaling for the treatment of gastric cancer

The Wnt signaling pathway is evolutionarily conserved, regulating both embryonic development and maintaining adult tissue homeostasis. Wnt signaling controls several fundamental cell functions, including proliferation, differentiation, migration, and stemness. It therefore plays an important role in the epithelial homeostasis and regeneration of the gastrointestinal tract. Often, both hypo- or hyper-activation of the pathway due to genetic, epigenetic, or receptor/ligand alterations are seen in many solid cancers, such as breast, colorectal, gastric, and prostate. Gastric cancer (GC) is the fourth commonest cause of cancer worldwide and is the second leading cause of cancer-related death annually. Although the number of new diagnoses has declined over recent decades, prognosis remains poor, with only 15% surviving to five years. Geographical differences in clinicopathological features are also apparent, with epidemiological and genetic studies revealing GC to be a highly heterogeneous disease with phenotypic diversity as a result of etiological factors. The molecular heterogeneity associated with GC dictates that a single ‘one size fits all’ approach to management is unlikely to be successful. Wnt pathway dysregulation has been observed in approximately 50% of GC tumors and may offer a novel therapeutic target for patients who would otherwise have a poor outcome. This mini review will highlight some recent discoveries involving Wnt signaling in GC

Exploring the Wnt pathway as a therapeutic target for prostate cancer

Aberrant activation of the Wnt pathway is emerging as a frequent event during prostate cancer that can facilitate tumor formation, progression, and therapeutic resistance. Recent discoveries indicate that targeting the Wnt pathway to treat prostate cancer may be efficacious. However, the functional consequence of activating the Wnt pathway during the different stages of prostate cancer progression remains unclear. Preclinical work investigating the efficacy of targeting Wnt signaling for the treatment of prostate cancer, both in primary and metastatic lesions, and improving our molecular understanding of treatment responses is crucial to identifying effective treatment strategies and biomarkers that help guide treatment decisions and improve patient care. In this review, we outline the type of genetic alterations that lead to activated Wnt signaling in prostate cancer, highlight the range of laboratory models used to study the role of Wnt genetic drivers in prostate cancer, and discuss new mechanistic insights into how the Wnt cascade facilitates prostate cancer growth, metastasis, and drug resistance

Frizzled-7 is required for Wnt signaling in gastric tumours with and without Apc mutations

A subset of patients with gastric cancer have mutations in genes that participate in or regulate Wnt signaling at the level of ligand (Wnt) receptor (Fzd) binding. Moreover, increased Fzd expression is associated with poor clinical outcome. Despite these findings, there are no in vivo studies investigating the potential of targeting Wnt receptors for treating gastric cancer, and the specific Wnt receptor transmitting oncogenic Wnt signaling in gastric cancer is unknown. Here, we use inhibitors of Wnt/Fzd (OMP-18R5/vantictumab) and conditional gene deletion to test the therapeutic potential of targeting Wnt signaling in preclinical models of intestinal-type gastric cancer and ex vivo organoid cultures. Pharmacologic targeting of Fzd inhibited the growth of gastric adenomas in vivo. We identified Fzd7 to be the predominant Wnt receptor responsible for transmitting Wnt signaling in human gastric cancer cells and mouse models of gastric cancer, whereby Fzd7-deficient cells were retained in gastric adenomas but were unable to respond to Wnt signals and consequently failed to proliferate. Genetic deletion of Fzd7 or treatment with vantictumab was sufficient to inhibit the growth of gastric adenomas with or without mutations to Apc. Vantictumab is currently in phase Ib clinical trials for advanced pancreatic, lung, and breast cancer. Our data extend the scope of patients that may benefit from this therapeutic approach as we demonstrate that this drug will be effective in treating patients with gastric cancer regardless of APC mutation status

Development of a low-seroprevalence, αvβ6 integrin-selective virotherapy based on human adenovirus type 10

Oncolytic virotherapies (OV) hold immense clinical potential. OV based on human adenoviruses (HAdV) derived from HAdV with naturally low rates of pre- existing immunity will be beneficial for future clinical translation. We generated a low- seroprevalence HAdV-D10 serotype vector incorporating an αvβ6 integrin-selective peptide, A20, to target αvβ6-positive tumor cell types. HAdV-D10 has limited natural tropism. Structural and biological studies of HAdV-D10 knob protein highlighted low-affinity engagement with native adenoviral receptors CAR and sialic acid. HAdV-D10 fails to engage blood coagulation factor X, potentially eliminating “off-target” hepatic sequestration in vivo. We engineered an A20 peptide that selectively binds αvβ6 integrin into the DG loop of HAdV-D10 fiber knob. Assays in αvβ6+ cancer cell lines demonstrated significantly increased transduction mediated by αvβ6-targeted variants compared with controls, confirmed microscopically. HAdV-D10.A20 resisted neutralization by neutralizing HAdV-C5 sera. Systemic delivery of HAdV-D10.A20 resulted in significantly increased GFP expression in BT20 tumors. Replication-competent HAdV-D10.A20 demonstrated αvβ6 integrin-selective cell killing in vitro and in vivo. HAdV-D10 possesses characteristics of a promising virotherapy, combining low seroprevalence, weak receptor interactions, and reduced off-target uptake. Incorporation of an αvβ6 integrin-selective peptide resulted in HAdV-D10.A20, with significant potential for clinical translation

The interaction of Wnt-11 and signalling cascades in prostate cancer

Prostate cancer (PCa) is the second most common cancer among the male population. Conventional therapies target androgen signalling, which drives tumour growth; however, they provide limited survival benefits for patients. It is essential, therefore, to develop a more specific biomarker than the current gold standard, PSA testing. The Wnt signalling pathway induces expression of target genes through cell surface receptors. A non-canonical member of this family, Wnt-11, is evolutionarily highly conserved and is normally expressed by various cells in the developing embryo, as well as in the heart, liver and skeletal muscle of adult humans. We comprehensively review several cell signalling pathways to explain how they interact with Wnt-11, demonstrating its use as a potential biomarker for PCa. Several studies have shown that the expression of Wnt-11 is associated with gastric, renal and colorectal adenocarcinomas and PCa. Moreover, Wnt-11 affects extracellular matrix composition and cytoskeletal rearrangement, and it is required for proliferation and/or survival during cell differentiation. It was found that PCa cell lines express high levels of Wnt-11, which allows differentiation of the epithelial prostate tumour cells to neuron-like (NE) cells. The NE cells produce additional factors that can cause regression after treatment. Accumulating evidence shows that Wnt-11 could be a potential biomarker in diagnosing PCa. Many studies have shown both non-canonical and canonical Wnts interact with several signalling cascades such as PKC, JNK, NF-κB, Rho, PKA and PI3K. In particular, evidence demonstrates Wnt-11 is involved in the progression of PCa, thus it could have the potential to become both a specific disease marker and an important therapeutic target

Identification of Pik3ca mutation as a genetic driver of prostate cancer that cooperates with Pten loss to accelerate progression and castration-resistant growth

Genetic alterations that potentiate PI3K signalling are frequent in prostate cancer, yet how different genetic drivers of the PI3K cascade contribute to prostate cancer is unclear. Here, we report PIK3CA mutation/amplification correlates with poor prostate cancer patient survival. To interrogate the requirement of different PI3K genetic drivers in prostate cancer, we employed a genetic approach to mutate Pik3ca in mouse prostate epithelium. We show Pik3caH1047R mutation causes p110-dependent invasive prostate carcinoma in-vivo. Furthermore, we report PIK3CA mutation and PTEN loss co-exist in prostate cancer patients, and can cooperate in-vivo to accelerate disease progression via AKT-mTORC1/2 hyperactivation. Contrasting single mutants that slowly acquire castration-resistant prostate cancer (CRPC), concomitant Pik3ca mutation and Pten loss caused de-novo CRPC. Thus, Pik3ca mutation and Pten deletion are not functionally redundant. Our findings indicate that PIK3CA mutation is an attractive prognostic indicator for prostate cancer that may cooperate with PTEN loss to facilitate CRPC in patients

Targeting Wnt Signaling for the Treatment of Gastric Cancer

The Wnt signaling pathway is evolutionarily conserved, regulating both embryonic development and maintaining adult tissue homeostasis. Wnt signaling controls several fundamental cell functions, including proliferation, differentiation, migration, and stemness. It therefore plays an important role in the epithelial homeostasis and regeneration of the gastrointestinal tract. Often, both hypo- or hyper-activation of the pathway due to genetic, epigenetic, or receptor/ligand alterations are seen in many solid cancers, such as breast, colorectal, gastric, and prostate. Gastric cancer (GC) is the fourth commonest cause of cancer worldwide and is the second leading cause of cancer-related death annually. Although the number of new diagnoses has declined over recent decades, prognosis remains poor, with only 15% surviving to five years. Geographical differences in clinicopathological features are also apparent, with epidemiological and genetic studies revealing GC to be a highly heterogeneous disease with phenotypic diversity as a result of etiological factors. The molecular heterogeneity associated with GC dictates that a single ‘one size fits all’ approach to management is unlikely to be successful. Wnt pathway dysregulation has been observed in approximately 50% of GC tumors and may offer a novel therapeutic target for patients who would otherwise have a poor outcome. This mini review will highlight some recent discoveries involving Wnt signaling in GC