The PI3K-AKT-mTOR pathway and prostate cancer: at the crossroads of AR, MAPK, and WNT signaling

Oncogenic activation of the phosphatidylinositol-3-kinase (PI3K), protein kinase B (PKB/AKT), and mammalian target of rapamycin (mTOR) pathway is a frequent event in prostate cancer that facilitates tumor formation, disease progression and therapeutic resistance. Recent discoveries indicate that the complex crosstalk between the PI3K-AKT-mTOR pathway and multiple interacting cell signaling cascades can further promote prostate cancer progression and influence the sensitivity of prostate cancer cells to PI3K-AKT-mTOR-targeted therapies being explored in the clinic, as well as standard treatment approaches such as androgen-deprivation therapy (ADT). However, the full extent of the PI3K-AKT-mTOR signaling network during prostate tumorigenesis, invasive progression and disease recurrence remains to be determined. In this review, we outline the emerging diversity of the genetic alterations that lead to activated PI3K-AKT-mTOR signaling in prostate cancer, and discuss new mechanistic insights into the interplay between the PI3K-AKT-mTOR pathway and several key interacting oncogenic signaling cascades that can cooperate to facilitate prostate cancer growth and drug-resistance, specifically the androgen receptor (AR), mitogen-activated protein kinase (MAPK), and WNT signaling cascades. Ultimately, deepening our understanding of the broader PI3K-AKT-mTOR signaling network is crucial to aid patient stratification for PI3K-AKT-mTOR pathway-directed therapies, and to discover new therapeutic approaches for prostate cancer that improve patient outcome

PTEN loss and activation of K-RAS and β-catenin cooperate to accelerate prostate tumourigenesis

Aberrant phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase (MAPK) and WNT signalling are emerging as key events in the multistep nature of prostate tumourigenesis and progression. Here, we report a compound prostate cancer murine model in which these signalling pathways cooperate to produce a more aggressive prostate cancer phenotype. Using Cre-LoxP technology and the probasin promoter, we combined the loss of Pten (Ptenfl/fl), to activate the PI3K signalling pathway, with either dominant stabilized β-catenin [Catnb+/lox(ex3)] or activated K-RAS (K-Ras+/V12) to aberrantly activate WNT and MAPK signalling, respectively. Synchronous activation of all three pathways (triple mutants) significantly reduced survival (median 96 days) as compared with double mutants [median: 140 days for Catnb+/lox(ex3)Ptenfl/fl; 182 days for Catnb+/lox(ex3)K-Ras+/V12; 238 days for Ptenfl/flK-Ras+/V12], and single mutants [median: 383 days for Catnb+/lox(ex3); 407 days for Ptenfl/fl], reflecting the accelerated tumourigenesis. Tumours followed a stepwise progression from mouse prostate intraepithelial neoplasia to invasive adenocarcinoma, similar to that seen in human disease. There was significantly elevated cellular proliferation, tumour growth and percentage of invasive adenocarcinoma in triple mutants as compared with double mutants and single mutants. Triple mutants showed not only activated AKT, extracellular-signal regulated kinase 1/2, and nuclear β-catenin, but also significantly elevated signalling through mechanistic target of rapamycin complex 1 (mTORC1). In summary, we show that combined deregulation of the PI3K, MAPK and WNT signalling pathways drives rapid progression of prostate tumourigenesis, and that deregulation of all three pathways results in tumours showing aberrant mTORC1 signalling. As mTORC1 signalling is emerging as a key driver of androgen deprivation therapy resistance, our findings are important for understanding the biology of therapy-resistant prostate cancer and identifying potential approaches to overcome this

CD200 ectodomain shedding into the tumor microenvironment leads to NK cell dysfunction and apoptosis

The basis of immune evasion, a hallmark of cancer, can differ even when cancers arise from one cell type such as in the human skin keratinocyte carcinomas: basal and squamous cell carcinoma. Here we showed that the basal cell carcinoma tumor initiating cell surface protein CD200, through ectodomain shedding, was responsible for the near absence of NK cells within the basal cell carcinoma tumor microenvironment. In situ, CD200 underwent ectodomain shedding by metalloproteinases MMP3 and MMP11, which released biologically active soluble CD200 into the basal cell carcinoma microenvironment. CD200 bound its cognate receptor on NK cells, to suppress MAPK pathway signaling that in turn blocked indirect (gamma interferon release) and direct cell killing. In addition, reduced ERK phosphorylation relinquished negative regulation of PPARγ regulated gene transcription and lead to membrane accumulation of the Fas/FADD death receptor and its ligand, FasL that resulted in activation-induced apoptosis. Blocking CD200 inhibition of MAPK or PPARγ signaling restored NK cell survival and tumor cell killing, with relevance to many cancer types. Our results thus uncover a paradigm for CD200 as a potentially novel and targetable NK cell specific immune checkpoint, which is responsible for NK cell associated poor outcomes in many cancers

Lkb1 Deficiency Alters Goblet and Paneth Cell Differentiation in the Small Intestine

The Lkb1 tumour suppressor is a multitasking kinase participating in a range of physiological processes. We have determined the impact of Lkb1 deficiency on intestinal homeostasis, particularly focussing on secretory cell differentiation and development since we observe strong expression of Lkb1 in normal small intestine Paneth and goblet cells. We crossed mice bearing an Lkb1 allele flanked with LoxP sites with those carrying a Cyp1a1-specific inducible Cre recombinase. Lkb1 was efficiently deleted from the epithelial cells of the mouse intestine after intraperitoneal injection of the inducing agent β-naphthoflavone. Bi-allelic loss of Lkb1 led to the perturbed development of Paneth and goblet cell lineages. These changes were characterised by the lack of Delta ligand expression in Lkb1-deficient secretory cells and a significant increase in the levels of the downstream Notch signalling effector Hes5 but not Hes1. Our data show that Lkb1 is required for the normal differentiation of secretory cell lineages within the intestine, and that Lkb1 deficiency modulates Notch signalling modulation in post-mitotic cells

Lkb1 and Pten Synergise to Suppress mTOR-Mediated Tumorigenesis and Epithelial-Mesenchymal Transition in the Mouse Bladder

The AKT/PI3K/mTOR pathway is frequently altered in a range of human tumours, including bladder cancer. Here we report the phenotype of mice characterised by deletion of two key players in mTOR regulation, Pten and Lkb1, in a range of tissues including the mouse urothelium. Despite widespread recombination within the range of epithelial tissues, the primary phenotype we observe is the rapid onset of bladder tumorigenesis, with median onset of approximately 100 days. Single deletion of either Pten or Lkb1 had no effect on bladder cell proliferation or tumour formation. However, simultaneous deletion of Lkb1 and Pten led to an upregulation of the mTOR pathway and the hypoxia marker GLUT1, increased bladder epithelial cell proliferation and ultimately tumorigenesis. Bladder tissue also exhibited characteristic features of epithelial-mesenchymal transition, with loss of the epithelial markers E-cadherin and the tight junction protein ZO-1, and increases in the mesenchymal marker vimentin as well as nuclear localization of epithelial-mesenchymal transition (EMT) regulator Snail. We show that these effects were all dependent upon mTOR activity, as rapamycin treatment blocked both EMT and tumorigenesis. Our data therefore establish clear synergy between Lkb1 and Pten in controlling the mTOR pathway within bladder epithelium, and show that loss of this control leads to the disturbance of epithelial structure, EMT and ultimately tumorigenesis

Brg1 loss is incompatible with Wnt-driven adenoma formation and improves animal survival upon Apc inactivation in the small intestine.

<p>A. Immunohistochemical analysis of β-catenin revealed numerous lesions with nuclear localisation of β-catenin in the small intestine of <i>AhCreER^T+Apc^fl/fl</i> and <i>AhCreER^T+Apc^fl/flBrg^fl/fl</i> mice 10 days post induction. Brg1 immunostaining of serial sections showed clusters of Brg1-deficient cells in <i>AhCreER^T+Apc^fl/flBrg^fl/fl</i> intestine, but failed to detect any overlap between lesions with nuclear β-catenin and Brg1 negative cells. B. Survival analysis demonstrated significantly increased survival probability of <i>AhCreER^T+Apc^fl/flBrg^fl/fl</i> mice (blue line, median survival 58 days) compared to <i>AhCreER^T+Apc^fl/fl</i> animals (green line, median survival 9 days). Log-rank test p<0.0001, n≥20. No control animals died within the timeframe of the experiment (n = 8). C. β-catenin immunostaining of the small intestinal epithelium of <i>AhCreER^T+Apc^fl/flBrg^fl/fl</i> mice 100 days post induction revealed numerous micro adenomas enclosed within distended villi. Staining with Brg1 antibody showed that all the micro adenomas retained Brg1 expression. Scale bars represent 100 µm (A) and 200 µm (B).</p

Brg1 Loss Attenuates Aberrant Wnt-Signalling and Prevents Wnt-Dependent Tumourigenesis in the Murine Small Intestine

<div><p>Tumourigenesis within the intestine is potently driven by deregulation of the Wnt pathway, a process epigenetically regulated by the chromatin remodelling factor Brg1. We aimed to investigate this interdependency in an <i>in vivo</i> setting and assess the viability of Brg1 as a potential therapeutic target. Using a range of transgenic approaches, we deleted <i>Brg1</i> in the context of Wnt-activated murine small intestinal epithelium. Pan-epithelial loss of Brg1 using <i>VillinCreER^T2</i> and <i>AhCreER^T</i> transgenes attenuated expression of Wnt target genes, including a subset of stem cell-specific genes and suppressed Wnt-driven tumourigenesis improving animal survival. A similar increase in survival was observed when Wnt activation and Brg1 loss were restricted to the Lgr5 expressing intestinal stem cell population. We propose a mechanism whereby Brg1 function is required for aberrant Wnt signalling and ultimately for the maintenance of the tumour initiating cell compartment, such that loss of Brg1 in an Apc-deficient context suppresses adenoma formation. Our results highlight potential therapeutic value of targeting Brg1 and serve as a proof of concept that targeting the cells of origin of cancer may be of therapeutic relevance.</p></div

Brg1 loss attenuates the effects of Apc deletion in the small intestinal epithelium.

<p>A. H&E, β-catenin and Brg1 staining of the small intestinal epithelium from control <i>VillinCreER^T2−</i>, <i>VillinCreER^T2+Apc^fl/fl</i> and <i>VillinCreER^T2+Apc^fl/flBrg^fl/fl</i> mice 4 days after high-dose induction revealed disturbed crypt architecture and nuclear localisation of β-catenin in both cohorts, as well as complete loss of Brg1 in <i>VillinCreER^T2+Apc^fl/flBrg^fl/fl</i> epithelium. Lysozyme immunostaining showed mis-localisation of Paneth cells in <i>VillinCreER^T2+Apc^fl/fl</i> epithelium, which was restored to normal by inactivation of Brg1. Scale bars represent 100 µm. B. Scoring of the crypt and villus length revealed no differences between double knock-out mice (black) and <i>VillinCreER^T2+Apc^fl/fl</i> animals (grey). Scoring of the cleaved Caspase3 positive cells and Ki67 positive cells detected a decrease in both apoptosis and proliferation in <i>VillinCreER^T2+Apc^fl/flBrg^fl/fl</i> mice compared to <i>VillinCreER^T2+Apc^fl/fl</i> animals. Data are shown as mean ± group's standard deviation for Caspase3 quantification and as mean ± pooled standard deviation otherwise, p value was calculated by means of t test not assuming equal variance for Caspase3 data and by means of one way ANOVA otherwise, p value was adjusted for multiple testing. For all comparisons n≥4. C. Analysis of cumulative frequency of Ki67 positive cells at each position along crypt-villus axis revealed expansion of the proliferative compartment in both double knock-out (green line) and <i>VillinCreER^T2+Apc^fl/fl</i> (orange line) mice compared to <i>VillinCreER^T2−</i> controls (blue line). This expansion was less pronounced in double knock-out mice compared to <i>VillinCreER^T2+Apc^fl/fl</i> animals. For all pair wise comparisons Kolmogorov-Smirnov test p<0.001, n = 4.</p