Aru Narendran

Tumour exosome integrins determine organotropic metastasis.

Ever since Stephen Paget’s 1889 hypothesis, metastatic organotropism has remained one of cancer’s greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis. Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.

Phase I Pharmacokinetic and Pharmacodynamic Study of 17-N-Allylamino-17-Demethoxygeldanamycin in Pediatric Patients with Recurrent or Refractory Solid Tumors: A Pediatric Oncology Experimental Therapeutics Investigators Consortium Study

Purpose: Heat shock protein 90 (Hsp90) is essential for the posttranslational control of many regulators of cell growth, differentiation, and apoptosis. 17-N-Allylamino-17-demethoxygeldanamycin (17-AAG) binds to Hsp90 and alters levels of proteins regulated by Hsp90. We conducted a phase I trial of 17-AAG in pediatric patients with recurrent or refractory neuroblastoma, Ewings sarcoma, osteosarcoma, and desmoplastic small round cell tumor to determine the maximum tolerated dose, define toxicity and pharmacokinetic profiles, and generate data about molecular target modulation. Experimental Design: Escalating doses of 17-AAG were administered i.v. over 1 to 2 h twice weekly for 2 weeks every 21 days until patients experienced disease progression or toxicity. harmacokinetic and pharmacodynamic studies were done during cycle 1. Results: Fifteen patients were enrolled onto dose levels between 150 and 360 mg/m2; 13 patients were evaluable for toxicity. The maximum tolerated dose was 270 mg/m2. DLTs were grade 3 transaminitis and hypoxia. Two patients with osteosarcoma and bulky pulmonary metastases died during cycle 1 and were not evaluable for toxicity. No objective responses were observed. 17-AAG pharmacokinetics in pediatric patients were linear; clearance and half-life were 21.6 ± 6.21 (mean ± SD) L/h/m2 and 2.6 ± 0.95 h, respectively. Posttherapy increases in levels of the inducible isoform of Hsp70, a marker of target modulation, were detected in peripheral blood mononuclear cells at all dose levels. Conclusion: 17-AAG was well tolerated at a dose of 270 mg/m2 administered twice weekly for 2 of 3 weeks. Caution should be used in treatment of patients with bulky pulmonary disease.

Survivin as a Preferential Target for Cancer Therapy

Cancer is typically a consequence of imbalance between cell death and proliferation in a way favorable to cell proliferation and survival. Most conventional cancer therapies are based on targeting rapidly growing cancerous cells to block growth or enhance cell death, thereby, restoring the balance between these processes. In many instances, malignancies that develop resistance to current treatment modalities, such as chemotherapy, immunotherapy, and radiotherapy often present the greatest challenge in subsequent management of the patient. Studies have shown that under normal circumstances, cells utilize different death mechanisms, such as apoptosis (programmed cell death), autophagy, mitotic catastrophe, and necrosis to maintain homeostasis and physiological integrity of the organism, but these processes often appear to be altered in cancer. Thus, in recent years developing various strategies for administration of cytotoxic chemotherapeutics in combination with apoptosis-sensitizing reagents is receiving more emphasis. Here, we review the properties of the anti-apoptotic protein, survivin, a member of the inhibitor of apoptosis protein (IAP) family and the clinical feasibility and anti-cancer potential of drugs targeting this protein. We also discuss some key points and concerns that should be taken into consideration while developing drugs that target apoptotic proteins, such as survivin.

Personalizing the treatment of pediatric medulloblastoma: Polo-like kinase 1 as a molecular target in high-risk children.

Medulloblastoma is the most common malignant brain tumor in children. This disease is heterogeneous and is composed of four subtypes of medulloblastoma [WNT, Sonic Hedgehog (SHH), Group 3, and Group 4]. An immediate goal is to identify novel molecular targets for the most aggressive forms of medulloblastoma. Polo-like kinase 1 (PLK1) is an oncogenic kinase that controls cell cycle and proliferation, making it a strong candidate for medulloblastoma treatment. In this study, pediatric medulloblastomas were subtyped in two patient cohorts (discovery cohort, n = 63 patients; validation cohort, n = 57 patients) using NanoString nCounter analysis and PLK1 mRNA was assessed. We determined that the SHH and Group 3 subtypes were independently associated with poor outcomes in children as was PLK1 using Cox regression analyses. Furthermore, we screened a library of 129 compounds in clinical trials using a model of pediatric medulloblastoma and determined that PLK1 inhibitors were the most promising class of agents against the growth of medulloblastoma. In patient-derived primary medulloblastoma isolates, the PLK1 small-molecule inhibitor BI2536 suppressed the self-renewal of cells with high PLK1 but not low PLK1 expression. PLK1 inhibition prevented medulloblastoma cell proliferation, self-renewal, cell-cycle progression, and induced apoptosis. In contrast, the growth of normal neural stem cells was unaffected by BI2536. Finally, BI2536 extended survival in medulloblastoma-bearing mice with efficacy comparable with Headstart, a standard-of-care chemotherapy regimen. We conclude that patients with medulloblastoma expressing high levels of PLK1 are at elevated risk. These preclinical studies pave the way for improving the treatment of medulloblastoma through PLK1 inhibition.

Polo‐Like Kinase 1 Inhibition Kills Glioblastoma Multiforme Brain Tumor Cells in Part Through Loss of SOX2 and Delays Tumor Progression in Mice

Glioblastoma multiforme (GBM) ranks among the deadliest types of cancer and given these new therapies are urgently needed. To identify molecular targets, we queried a microarray profiling 467 human GBMs and discovered that polo‐like kinase 1 (PLK1) was highly expressed in these tumors and that it clustered with the proliferative subtype. Patients with PLK1‐high tumors were more likely to die from their disease suggesting that current therapies are inactive against such tumors. This prompted us to examine its expression in brain tumor initiating cells (BTICs) given their association with treatment failure. BTICs isolated from patients expressed 110‐470 times more PLK1 than normal human astrocytes. Moreover, BTICs rely on PLK1 for survival because the PLK1 inhibitor BI2536 inhibited their growth in tumorsphere cultures. PLK1 inhibition suppressed growth, caused G2/M arrest, induced apoptosis, and reduced the expression of SOX2, a marker of neural stem cells, in SF188 cells. Consistent with SOX2 inhibition, the loss of PLK1 activity caused the cells to differentiate based on elevated levels of glial fibrillary acidic protein and changes in cellular morphology. We then knocked glial fibrillary acidic protein (GFAP) down SOX2 with siRNA and showed that it too inhibited cell growth and induced cell death. Likewise, in U251 cells, PLK1 inhibition suppressed cell growth, downregulated SOX2, and induced cell death. Furthermore, BI2536 delayed tumor growth of U251 cells in an orthotopic brain tumor model, demonstrating that the drug is active against GBM. In conclusion, PLK1 level is elevated in GBM and its inhibition restricts the growth of brain cancer cells. STEM CELLS2012;30:1064–1075

Adenovirus-mediated p53 gene therapy in osteosarcoma cell lines: sensitization to cisplatin and doxorubicin

The poor prognosis for patients with metastatic osteosarcoma (OS) indicates that new therapeutic options should be explored. Studies with adenoviral-mediated p53 gene transfer have been conducted in many cancer types including cervical, ovarian, prostatic and head and neck tumors. However, limited work has been carried out with pediatric cancers, including OS. Using three viral constructs containing cDNA for wild-type p53, mutant p53 (Cys135Ser) and lacZ, we studied the effect of adenoviral-mediated gene therapy in four OS cell lines: Saos-2 (p53−/−), HOS (R156P), KHOS/NP (R156P) and MNNG (R156P, F270L). We demonstrated that the virus efficiently enters the cells using the β-galactosidase assay. Using the MTT assay, we have shown a dose-dependent decrease in cell viability 72 h post-treatment that occurs with Ad-wtp53 but not with Ad-mutp53. We have also shown that treatment with Ad-wtp53 significantly increases sensitivity of the cell lines to cisplatin and doxorubicin, chemotherapeutic agents commonly used in the treatment of OS. Our results indicate that restoration of wt p53 function in OS cells provides a basis for novel approaches to treatment of this disease.

Adenovirus-mediated p53 gene therapy in pediatric soft-tissue sarcoma cell lines: sensitization to cisplatin and doxorubicin.

Sarcomas, or tumors of connective tissue, represent roughly 20% of childhood cancers. Although the cure rate for sarcomas in general has significantly improved in the last 10 years, there continue to be subgroups that are difficult to treat. High-grade or metastatic soft-tissue sarcomas and rhabdomyosarcomas (RMS) of the extremities remain therapeutic challenges and their prognosis is often poor. The future of sarcoma therapy will likely include molecular approaches including gene/protein expression profiling and gene-based therapy. Most sarcomas harbor defects in the p53 or pRb pathways. The tumor suppressor p53 is central to regulation of cell growth and tumor suppression and restoring wild-type p53 function in pediatric sarcomas may be of therapeutic benefit. Studies with adenoviral-mediated p53 gene transfer have been conducted in many cancer types including cervical, ovarian, prostatic and head and neck tumors. Studies of this approach, however, remain limited in pediatric cancers, including sarcomas. Using three viral constructs containing cDNA for wild-type p53, mutant p53 (C135S) and lacZ, we studied the effect of adenoviral-mediated gene therapy in four pediatric sarcoma cell lines, RD and Rh4 (RMS), Rh1 (Ewings sarcoma) and A204 (undifferentiated sarcoma). Using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, we have shown a dose-dependent decrease in cell viability 72 h post-treatment that occurs with Ad-wtp53 but not with Ad-mutp53. Cells treated with Ad-wtp53 show upregulation of the p53 downstream targets, p21CIP1/WAF1 and bax. Growth curves demonstrate suppression of cell growth over a period of 4 days and cells treated with Ad-wtp53 demonstrate a significant increase in sensitivity to the chemotherapeutic agents, cisplatin and doxorubicin. Our results indicate that restoration of wild-type p53 function in pediatric sarcoma cells could provide a basis for novel approaches to treatment of this disease.

Mutant p53 in bone marrow stromal cells increases VEGF expression and supports leukemia cell growth

OBJECTIVE Genetic alterations, including p53 mutations, have been identified in the stroma of solid tumors and are thought be involved in the induction of tumor growth and metastasis. We tested the hypothesis that somatic molecular alterations in bone marrow stromal cells provide a favorable growth environment for leukemic cells. MATERIALS AND METHODS We established an in vitro model consisting of stroma expressing mutant p53 (Cys135Ser) to study its ability to support growth of cells from a pre-B acute lymphoblastic leukemia (ALL) cell line. Normal and leukemic bone marrow stromal cells were screened for p53 mutations by mutant-specific ELISA, SSCP, and direct sequencing. Secretion of vascular endothelial growth factor (VEGF) was measured by quantitative ELISA. RESULTS Transfection of stromal cells with mutant p53 increased synthesis of VEGF and supported the growth of leukemic cells. An ELISA-based assay suggested the occurrence of in vivo p53 alterations in bone marrow stromal cells from 2 of 12 ALL patients screened. Direct sequencing of one of these samples revealed a somatic heterozygous p53 gene mutation (Asp49His). This sample secreted more VEGF and provided increased growth support to leukemic cells. The ability of Asp 49His-p53 to increase the expression of VEGF was confirmed with transfection experiments in a p53-null cell line. CONCLUSION Our findings indicate that genetic alterations, such as p53 mutations, in stromal cells can increase stromal-derived support of leukemia growth. Increased synthesis of pro-angiogenic cytokines, such as VEGF, may constitute one possible pathway by which this process is mediated.

CHARACTERIZATION OF BONE MARROW STROMAL ABNORMALITIES IN A PATIENT WITH CONSTITUTIONAL TRISOMY 8 MOSAICISM AND MYELODYSPLASTIC SYNDROME

The development of myeloid leukemias and myelodysplastic syndrome (MDS) is common in children with trisomy 8 mosaicism. However, the mechanisms by which the presence of an additional chromosome 8 translates to an increased risk of leukemias and MDS is currently unknown. The authors describe the analysis of stromal cells from a pediatric MDS patient with constitutional trisomy 8. Patient and control marrow stromal cells were analyzed for alterations in cytokine production. Clonogenic assays were used to examine stromal support for hematopoiesis. The interplay between leukemia cells and stroma was studied by co-culture experiments. The results indicate that stromal cell function in this patient was seriously altered in favor of progenitor cell proliferation and expansion. This indicates that constitutional trisomy 8 in stromal cells plays a critical role in the pathogenesis of MDS.

A novel C19MC amplified cell line links Lin28/let-7 to mTOR signaling in embryonal tumor with multilayered rosettes

BACKGROUND Embryonal tumor with multilayered rosettes (ETMR) is an aggressive central nervous system primitive neuroectodermal tumor (CNS-PNET) variant. ETMRs have distinctive histology, amplification of the chromosome 19 microRNA cluster (C19MC) at chr19q13.41-42, expression of the RNA binding protein Lin28, and dismal prognosis. Functional and therapeutic studies of ETMR have been limited by a lack of model systems. METHODS We have established a first cell line, BT183, from a case of ETMR and characterized its molecular and cellular features. LIN28 knockdown was performed in BT183 to examine the potential role of Lin28 in regulating signaling pathway gene expression in ETMR. Cell line findings were corroborated with immunohistochemical studies in ETMR tissues. A drug screen of 73 compounds was performed to identify potential therapeutic targets. RESULTS The BT183 line maintains C19MC amplification, expresses C19MC-encoded microRNAs, and is tumor initiating. ETMRs, including BT183, have high LIN28 expression and low let-7 miRNA expression, and show evidence of mTOR pathway activation. LIN28 knockdown increases let-7 expression and decreases expression of IGF/PI3K/mTOR pathway components. Pharmacologic inhibition of the mTOR pathway reduces BT183 cell viability. CONCLUSIONS BT183 retains key genetic and histologic features of ETMR. In ETMR, Lin28 is not only a diagnostic marker but also a regulator of genes involved in growth and metabolism. Our findings indicate that inhibitors of the IGF/PI3K/mTOR pathway may be promising novel therapies for these fatal embryonal tumors. As the first patient-derived cell line of these rare tumors, BT183 is an important, unique reagent for investigating ETMR biology and therapeutics.