Soft tissue sarcoma: biology and therapeutic correlates

Soft tissue sarcomas (STS) comprise a heterogenenous group of greater than 50 malignancies of putative mesenchymal cell origin and as such they may arise in diverse tissue types in various anatomical locations throughout the whole body. Collectively they account for approximately 1% of all human malignancies yet have a spectrum of aggressive behaviours amongst their subtypes. They thus pose a particular challenge to manage and remain an under investigated group of cancers with no generally applicable new therapies in the past 40 years and an overall 5-year survival rate that remains stagnant at around 50%. From September 2000 to July 2006 I undertook a full time post-doctoral level research fellowship at the MD Anderson Cancer Center, Houston, Texas, USA in the department of Surgical Oncology to investigate the biology of soft tissue sarcoma and test novel anti- sarcoma adenovirus-based therapy in the preclinical nude rat model of isolated limb perfusion against human sarcoma xenografts. This work, in collaboration with colleagues as indicated herein, led to a number of publications in the scientific literature furthering our understanding of the malignant phenotype of sarcoma and reported preclinical studies with wild-type p53, in a replication deficient adenovirus vector, and oncolytic adenoviruses administered by isolated limb perfusion. Additional collaborative and pioneering preclinical studies reported the molecular imaging of sarcoma response to systemically delivered therapeutic phage RGD-4c AAVP. Doxorubicin chemotherapy is the single most active broadly applicable anti-sarcoma chemotherapeutic yet only has an approximate 30% overall response rate with additional breakthrough tumour progression and recurrence after initial chemo-responsiveness further problematic features in STS management. Doxorubicin is a substrate for the multi- drug resistance (mdr) gene product p-glycoprotein drug efflux pump and exerts its main mode of action by induction of DNA double-strand breaks during the S-phase of the cell cycle. Two papers in my thesis characterise different aspects of chemoresistance in sarcoma. The first shows that wild-type p53 suppresses Protein Kinase Calpha (PKCα) phosphorylation (and activation) of p-glycoprotein by transcriptional repression of PKCα through a Sp-1 transcription factor binding site in its -244/-234 promoter region. The second paper demonstrates that Rad51 (a central mediator of homologous recombination repair of double strand breaks) has elevated levels in sarcoma and particularly in the S- G2 phase of the cell cycle. Suppression of Rad51 with small interfering RNA in sarcoma cell culture led to doxorubicin chemosensitisation. Reintroduction of wild-type p53 into STS cell lines resulted in decreased Rad51 protein and mRNA expression via transcriptional repression of the Rad51 promoter through increased AP-2 binding. In light of poor response rates to chemotherapy, escape from local control portends a poor prognosis for patients with sarcoma. Two papers in my thesis characterise aspects of sarcoma angiogenesis, invasion and metastasis. Human sarcoma samples were found to have high levels of matrix metalloproteinase-9 (MMP-9) with expression levels that correlated with p53 mutational status. MMP-9 is known to degrade extracellular collagen, contribute to the control of the angiogenic switch necessary in primary tumour progression and facilitate invasion and metastasis. Reconstitution of wild-type p53 function led to decreased levels of MMP-9 protein and mRNA as well as zymography-assessed MMP-9 proteolytic activity and decreased tumour cell invasiveness. Reintroduction of wild-type p53 into human sarcoma xenografts in-vivo decreased tumour growth and MMP-9 protein expression. Wild-type p53 was found to suppress mmp-9 transcription via decreased binding of NF-κB to its -607/-595 mmp-9 promoter element. Studies on the role of the VEGF165 in sarcoma found that sarcoma cells stably transfected with VEGF165 formed more aggressive xenografted tumours with increased vascularity, growth rate, metastasis, and resistance to chemotherapy. Use of the anti-VEGFR2 antibody DC101 enhanced doxorubicin sensitivity at sub-conventional dosing, inhibited tumour growth, decreased development of metastases, and reduced tumour micro-vessel density while increasing the vessel maturation index. These effects were explained primarily through effects on endothelial cells (e.c.s), rather than the tumour cells per se, where DC101 induced e.c. sensitivity to doxorubicin and suppressed e.c. production of MMPs. The p53 tumour suppressor pathway is the most frequently mutated pathway in sarcoma. Recapitulation of wild-type p53 function in sarcoma exerts a number of anti-cancer outcomes such as growth arrest, resensitisation to chemotherapy, suppression of invasion, and attenuation of angiogenesis. Using a modified nude rat-human sarcoma xenograft model for isolated limb perfusion (ILP) delivery of wild-type p53 in a replication deficient adenovirus vector I showed that functionally competent wild-type p53 could be delivered to and detected in human leiomyosarcoma xenografts confirming preclinical feasibility - although not efficacious due to low transgene expression. Viral fibre modification to express the RGD tripeptide motif led to greater viral uptake by sarcoma cells in vitro (transductional targeting) and changing the transgene promoter to a response element active in cells with active telomerase expression restricted the transgene expression to the tumour intracellular environment (transcriptional targeting). Delivery of the fibre-modified, selectively replication proficient oncolytic adenovirus Ad.hTC.GFP/ E1a.RGD by ILP demonstrated a more robust, and tumour-restricted, transgene expression with evidence of anti-sarcoma effect confirmed microscopically. Collaborative studies using the fibre modified phage RGD-4C AAVP confirmed that systemic delivery specifically, efficiently, and repeatedly targets human sarcoma xenografts, binds to αv integrins in tumours, and demonstrates a durable, though heterogeneous, transgene expression of 1-4 weeks. Incorporation of the Herpes Simplex Virus thymidine kinase (HSVtk) transgene into RGD-4C AAVP permitted CT-PET spatial and temporal molecular imaging in vivo of transgene expression and allowed quantification of tumour metabolic activity both before and after interval administration of a systemic cytotoxic with predictable and measurable response to treatment before becoming apparent clinically. These papers further the medical and scientific community’s understanding of the biology of soft tissue sarcoma and report preclinical studies with novel and promising anti- sarcoma therapeutics.

The role of microRNA 21 in the development of in-stent restenosis

Coronary heart disease is a major cause of morbidity and mortality worldwide. Percutaneous coronary intervention (PCI) has become the most widely used method of coronary artery revascularisation. The use of stents to hold open atherosclerosis induced arterial narrowing has significantly reduced elastic recoil and acute vessel occlusion following balloon angioplasty. However, bare metal stents have been associated with in-stent restenosis attributed to vascular smooth muscle cell (VSMC) hyperplasia and excessive neointimal formation. The resultant luminal renarrowing may manifest clinically with the return of symptoms such as chest pain or shortness of breath. The development of drug eluting stents has significantly reduced the incidence of in-stent restenosis (ISR). Unfortunately the antiproliferative medications used not only inhibit VSMC proliferation but also re-endothelialisation of the stented vessel. In addition, the drug impregnated polymer coating has been associated with a chronic inflammatory response within the vessel wall predisposing patients to stent thrombosis. Thus the identification of novel therapies which promote vessel healing without excessive proliferative or inflammatory response may improve long term outcome and reduce the need for repeated revascularisation. MicroRNAs (miRs) are short (18-25 nucleotide) non-coding RNAs acting to regulate gene expression. By binding to the 3’untranslated region of mRNA they act to fine tune gene expression either by mRNA degradation or translational repression. Originally identified in coordinating tissue development microRNAs have also been shown to play important roles coordinating the inflammatory response and in numerous cardiovascular diseases. MiR-21 has been identified in human atherosclerotic plaques, arteriosclerosis obliterans and abdominal aortic aneurysms. In addition, its up regulation has been documented in preclinical models of vascular injury. This study sought to identify the role of miR-21 in the development of ISR. Utilising a small animal model of stenting and in vitro techniques, we sought to investigate its influence upon VSMC and immune cell response following stenting. 19 The refinement of a murine stenting model within the Baker laboratory and the electrochemical dissolution of the metal stent from within harvested vascular tissues significantly improved the ability to perform detailed histological analysis. In addition, identification of miRNAs using in situ hybridisation was achieved for the first time within stented tissue. Neointimal formation and ISR was significantly reduced in mice in which miR-21 had been genetically deleted. In addition, neointimal composition was found to be altered in miR-21 KO mice with reductions in VSMC and elastin content demonstrated. Importantly, no difference in re-endothelialisation was observed. In vitro analysis demonstrated that VSMCs from miR-21 KO mice had both reduced proliferative and migratory capacity following platelet derived growth factor stimulation. Molecular analysis revealed that these differences may, at least in part, be due to de-repression of programmed cell death 4 (PDCD4). PDCD4 is a known miR-21 target within VSMCs implicated in the suppression of proliferation and promotion of apoptosis. Unfortunately, initial attempts at antimiR mediated knockdown of miR-21 in vivo, failed to produce a similar change in the suppression of ISR. Furthermore, a significant alteration in macrophage polarisation state within the neointima of miR-21 WT and KO mice was noted. Immunohistochemical staining revealed a preponderance of anti-inflammatory M2 macrophages in KO mice. Analysis of bone marrow derived macrophages from miR-21 KO mice demonstrated an increased level of the peroxisome proliferation activating receptor-γ (PPARγ) which facilitates M2 polarisation. Importantly, significant alterations in numerous pro-inflammatory cytokines, which also have mitogenic effects, were also found following genetic deletion of miR-21. In Summary, this is the first study to look at miRs in the development of ISR. MiR-21 plays an important role in the development of ISR by influencing the proliferative response of VSMCs and modulating the immune response following stent deployment. Further attempts to modulate miR-21 expression following PCI may reduce ISR and the need for repeat revascularisation while also reducing the risk of stent thrombosis.

Using induced pluripotent stem cells (IPSCS) as a replacement for in vivo models to screen novel therapies in chronic myeloid leukaemia (CML)

Tyrpsine kinase inhibitors (TKIs) effectively target progenitors and mature leukaemic cells but prove less effective at eliminating leukaemic stem cells (LSCs) in patients with chronic myeloid leukaemia (CML). Several reports indicate that the TGFβ superfamily pathway is important for LSC survival and quiescence. We conducted extensive microarray analyses to compare expression patterns in normal haemopoietic stem cells (HSC) and progenitors with CML LSC and progenitor populations in chronic phase (CP), accelerated phase (AP) and blast crisis (BC) CML. The BMP/SMAD pathway and downstream signalling molecules were identified as significantly deregulated in all three phases of CML. The changes observed could potentiate altered autocrine signalling, as BMP2, BMP4 (p<0.05), and ACTIVIN A (p<0.001) were all down regulated, whereas BMP7, BMP10 and TGFβ (p<0.05) were up regulated in CP. This was accompanied by up regulation of BMPRI (p<0.05) and downstream SMADs (p<0.005). Interestingly, as CML progressed, the profile altered, with BC patients showing significant over-expression of ACTIVIN A and its receptor ACVR1C. To further characterise the BMP pathway and identify potential candidate biomarkers within a larger cohort, expression analysis of 42 genes in 60 newly diagnosed CP CML patient samples, enrolled on a phase III clinical trial (www.spirit-cml.org) with greater than 12 months follow-up data on their response to TKI was performed. Analysis revealed that the pathway was highly deregulated, with no clear distinction when patients were stratified into good, intermediate and poor response to treatment. One of the major issues in developing new treatments to target LSCs is the ability to test small molecule inhibitors effectively as it is difficult to obtain sufficient LSCs from primary patient material. Using reprogramming technologies, we generated induced pluripotent stem cells (iPSCs) from CP CML patients and normal donors. CML- and normal-derived iPSCs were differentiated along the mesodermal axis to generate haemopoietic and endothelial precursors (haemangioblasts). IPSC-derived haemangioblasts exhibited sensitivity to TKI treatment with increased apoptosis and reduction in the phosphorylation of downstream target proteins. 4 Dual inhibition studies were performed using BMP pathway inhibitors in combination with TKI on CML cell lines, primary cells and patient derived iPSCs. Results indicate that they act synergistically to target CML cells both in the presence and absence of BMP4 ligand. Inhibition resulted in decreased proliferation, irreversible cell cycle arrest, increased apoptosis, reduced haemopoietic colony formation, altered gene expression pattern, reduction in self-renewal and a significant reduction in the phosphorylation of downstream target proteins. These changes offer a therapeutic window in CML, with intervention using BMP inhibitors in combination with TKI having the potential to prevent LSC self-renewal and improve outcome for patients. By successfully developing and validating iPSCs for CML drug screening we hope to substantially reduce the reliance on animal models for early preclinical drug screening in leukaemia.