A novel, angiogenesis-independent role for pericytes in ovarian cancer progression and metastasis

Cancer-associated fibroblasts (CAFs) are a heterogeneous population of cells known for their pro-tumourigenic effect in the microenvironment of solid tumours. One of their major sources is bone marrow derived mesenchymal stem cells (BM MSCs), a population with a demonstrated ability to infiltrate the site of the tumour and engage in paracrine cross-talk with the tumour cells. However, the role of a locally resident perivascular MSC population, i.e. pericytes, has not been explored in this context. Pericytes are contractile cells located around microvessels and are known for their prominent role in maintenance and stabilisation of the blood vasculature in homeostasis, and during tumour angiogenesis. However, previous data from our laboratory has shown that pericytes have the ability to promote the growth of normal epithelial cells, independent of angiogenesis. In addition, interrogation of a gene expression dataset of 215 serous ovarian cancer patients revealed that patients carrying a pericyte-specific gene expression signature bore a significantly higher risk of relapse and a lower overall chance of survival. This indicated that pericyte activity is a strong predictor of cancer recurrence and mortality. Further, this high-risk patient group was distinct from that identified by an angiogenic signature. Therefore, this thesis work was undertaken with the hypothesis that pericytes have a direct, pro-proliferative role in promoting tumour growth and metastasis, independent of their known roles in regulating angiogenesis. In the work presented here, co-injection of exogenous human pericytes with tumour cells resulted in increased tumour growth and metastasis in a xenograft model of ovarian cancer. Importantly, this effect was not associated with a concomitant increase in the extent and distribution of blood vessels, or alterations in other crucial angiogenic processes. Further, histological analyses of the xenograft tumours revealed increased tumour invasion leading to epithelial mesenchymal transition (EMT) in the presence of pericytes. Interestingly, co-injection of pericytes led to an increased infiltration of cells expressing alpha SMA to non-blood vessel-associated sites within xenograft tumours - a notable feature in tumor tissue from patients with relatively earlier recurrence of disease. In consistence with this observation, alpha SMA expression was predictive for relapse in ovarian cancer patients. Further in silico analysis revealed that extracellular matrix (ECM) remodelling and focal adhesion were the primary pathways overexpressed in ovarian cancer patients with early recurrence of disease. Together, findings from this study led to the conclusion that the MSC-like pericytes, when dissociated from blood vessels or mislocalised at non-vascular sites, can directly engage in paracrine crosstalk with tumour cells, resulting in increased proliferation, invasion and metastasis, without affecting angiogenesis. Thus, this study is the first clinical and experimental evidence for a novel, angiogenesis-independent, CAF-like role for pericytes in the ovarian tumour microenvironment and presents the possibility of pericyte-specific genes being used at the protein level as prognostic markers for ovarian cancer progression and survival. Further studies focussing on deciphering the molecular mechanisms through which pericytes exert their pro-proliferative effects in the tumour microenvironment are required for better understanding of their biology and precise role in cancer pathogenesis

Pericytes promote malignant ovarian cancer progression in mice and predict poor prognosis in serous ovarian cancer patients

Purpose: The aim of this study was to investigate the role of pericytes in regulating malignant ovarian cancer progression. Experimental Design: The pericyte mRNA signature was used to interrogate ovarian cancer patient datasets to determine its prognostic value for recurrence and mortality. Xenograft models of ovarian cancer were used to determine if co-injection with pericytes affected tumor growth rate and metastasis, whereas co-culture models were utilized to investigate the direct effect of pericytes on ovarian cancer cells. Pericyte markers were used to stain patient tissue samples to ascertain their use in prognosis. Results: Interrogation of two serous ovarian cancer patient datasets [the Australian Ovarian Cancer Study, n = 215; and the NCI TCGA (The Cancer Genome Atlas), n = 408] showed that a high pericyte score is highly predictive for poor patient prognosis. Co-injection of ovarian cancer (OVCAR-5 & -8) cells with pericytes in a xenograft model resulted in accelerated ovarian tumor growth, and aggressive metastases, without altering tumor vasculature. Pericyte co-culture in vitro promoted ovarian cancer cell proliferation and invasion. High αSMA protein levels in patient tissue microarrays were correlated with more aggressive disease and earlier recurrence. Conclusions: High pericyte score provides the best means to date of identifying patients with ovarian cancer at high risk of rapid relapse and mortality (mean progression-free survival time < 9 months). The stroma contains rare yet extremely potent locally resident mesenchymal stem cells—a subset of “cancer-associated fibroblasts” that promote aggressive tumor growth and metastatic dissemination, underlying the prognostic capacity of a high pericyte score to strongly predict earlier relapse and mortality

BET inhibitor resistance emerges from leukaemia stem cells

Bromodomain and extra terminal protein (BET) inhibitors are first-in-class targeted therapies that deliver a new therapeutic opportunity by directly targeting bromodomain proteins that bind acetylated chromatin marks. Early clinical trials have shown promise, especially in acute myeloid leukaemia, and therefore the evaluation of resistance mechanisms is crucial to optimize the clinical efficacy of these drugs. Here we use primary mouse haematopoietic stem and progenitor cells immortalized with the fusion protein MLL-AF9 to generate several single-cell clones that demonstrate resistance, in vitro and in vivo, to the prototypical BET inhibitor, I-BET. Resistance to I-BET confers cross-resistance to chemically distinct BET inhibitors such as JQ1, as well as resistance to genetic knockdown of BET proteins. Resistance is not mediated through increased drug efflux or metabolism, but is shown to emerge from leukaemia stem cells both ex vivo and in vivo. Chromatin-bound BRD4 is globally reduced in resistant cells, whereas the expression of key target genes such as Myc remains unaltered, highlighting the existence of alternative mechanisms to regulate transcription. We demonstrate that resistance to BET inhibitors, in human and mouse leukaemia cells, is in part a consequence of increased Wnt/β-catenin signalling, and negative regulation of this pathway results in restoration of sensitivity to I-BET in vitro and in vivo. Together, these findings provide new insights into the biology of acute myeloid leukaemia, highlight potential therapeutic limitations of BET inhibitors, and identify strategies that may enhance the clinical utility of these unique targeted therapies