Exploring transcriptional regulators Ref-1 and STAT3 as therapeutic targets in malignant peripheral nerve sheath tumours.

BACKGROUND: MPNST is a rare soft-tissue sarcoma that can arise from patients with NF1. Existing chemotherapeutic and targeted agents have been unsuccessful in MPNST treatment, and recent findings implicate STAT3 and HIF1-α in driving MPNST. The DNA-binding and transcriptional activity of both STAT3 and HIF1-α is regulated by Redox factor-1 (Ref-1) redox function. A first-generation Ref-1 inhibitor, APX3330, is being tested in cancer clinical trials and could be applied to MPNST. METHODS: We characterised Ref-1 and p-STAT3 expression in various MPNST models. Tumour growth, as well as biomarkers of apoptosis and signalling pathways, were measured by qPCR and western blot following treatment with inhibitors of Ref-1 or STAT3. RESULTS: MPNSTs from Nf1-Arfflox/floxPostnCre mice exhibit significantly increased positivity of p-STAT3 and Ref-1 expression when malignant transformation occurs. Inhibition of Ref-1 or STAT3 impairs MPNST growth in vitro and in vivo and induces apoptosis. Genes highly expressed in MPNST patients are downregulated following inhibition of Ref-1 or STAT3. Several biomarkers downstream of Ref-1 or STAT3 were also downregulated following Ref-1 or STAT3 inhibition. CONCLUSIONS: Our findings implicate a unique therapeutic approach to target important MPNST signalling nodes in sarcomas using new first-in-class small molecules for potential translation to the clinic.

Neurofibromatosis type 1: Fundamental insights into cell signalling and cancer.

Neurofibromatosis type 1 (NF1) is an autosomal dominant tumour predisposition syndrome that is caused through loss of function mutations of a tumour suppressor gene called Neurofibromin 1. Therapeutic options are currently limited for NF1-associated tumours, where treatment is often restricted to complete surgical resection with clear margins. Herein, we discuss the multifunctional tumour suppressive role of neurofibromin, which is classically known as a GTPase activating protein (GAP) towards the RAS small GTPase. While neurofibromin inhibits proliferative growth through blockade of RAS-mediated signal transduction, neurofibromin should also be considered as a modulator of cell motility and cell adhesion. Through interfacing with the cytoskeleton and membrane structures, neurofibromin acts as a negative regulator of RHO/ROCK signalling pathways involved in cytoskeletal dynamics that are instrumental in proper neuronal development. In the context of cancer, the loss of normal function of neurofibromin via genetic mutation results in heightened cell proliferation and migration, predisposing NF1 patients to cancer. Malignant Peripheral Nerve Sheath Tumours (MPNSTs) can develop from benign neurofibromas and are the main cause of death amongst NF1 patients. Through recent research on MPNSTs, we have gained insight into the key molecular events that drive their malignancy. Advances regarding malignant drivers involved in cell migration, cell invasion and angiogenic signalling are discussed in this review, where these findings will likely influence future therapies for both NF1 and related sporadic cancers.

Evaluation of copy number variation and gene expression in neurofibromatosis type-1-associated malignant peripheral nerve sheath tumours.

BACKGROUND: Neurofibromatosis type-1 (NF1) is a complex neurogenetic disorder characterised by the development of benign and malignant tumours of the peripheral nerve sheath (MPNSTs). Whilst biallelic NF1 gene inactivation contributes to benign tumour formation, additional cellular changes in gene structure and/or expression are required to induce malignant transformation. Although few molecular profiling studies have been performed on the process of progression of pre-existing plexiform neurofibromas to MPNSTs, the integrated analysis of copy number alterations (CNAs) and gene expression is likely to be key to understanding the molecular mechanisms underlying NF1-MPNST tumorigenesis. In a pilot study, we employed this approach to identify genes differentially expressed between benign and malignant NF1 tumours. RESULTS: SPP1 (osteopontin) was the most differentially expressed gene (85-fold increase in expression), compared to benign plexiform neurofibromas. Short hairpin RNA (shRNA) knockdown of SPP1 in NF1-MPNST cells reduced tumour spheroid size, wound healing and invasion in four different MPNST cell lines. Seventy-six genes were found to exhibit concordance between CNA and gene expression level. CONCLUSIONS: Pathway analysis of these genes suggested that glutathione metabolism and Wnt signalling may be specifically involved in NF1-MPNST development. SPP1 is associated with malignant transformation in NF1-associated MPNSTs and could prove to be an important target for therapeutic intervention.

Drug Inhibition of Redox Factor-1 Restores Hypoxia-Driven Changes in Tuberous Sclerosis Complex 2 Deficient Cells.

Therapies with the mechanistic target of rapamycin complex 1 (mTORC1) inhibitors are not fully curative for tuberous sclerosis complex (TSC) patients. Here, we propose that some mTORC1-independent disease facets of TSC involve signaling through redox factor-1 (Ref-1). Ref-1 possesses a redox signaling activity that stimulates the transcriptional activity of STAT3, NF-kB, and HIF-1α, which are involved in inflammation, proliferation, angiogenesis, and hypoxia, respectively. Here, we demonstrate that redox signaling through Ref-1 contributes to metabolic transformation and tumor growth in TSC cell model systems. In TSC2-deficient cells, the clinically viable Ref-1 inhibitor APX3330 was effective at blocking the hyperactivity of STAT3, NF-kB, and HIF-1α. While Ref-1 inhibitors do not inhibit mTORC1, they potently block cell invasion and vasculature mimicry. Of interest, we show that cell invasion and vasculature mimicry linked to Ref-1 redox signaling are not blocked by mTORC1 inhibitors. Metabolic profiling revealed that Ref-1 inhibitors alter metabolites associated with the glutathione antioxidant pathway as well as metabolites that are heavily dysregulated in TSC2-deficient cells involved in redox homeostasis. Therefore, this work presents Ref-1 and associated redox-regulated transcription factors such as STAT3, NF-kB, and HIF-1α as potential therapeutic targets to treat TSC, where targeting these components would likely have additional benefits compared to using mTORC1 inhibitors alone.

Oncogenic Signalling through Mechanistic Target of Rapamycin (mTOR): A Driver of Metabolic Transformation and Cancer Progression.

Throughout the years, research into signalling pathways involved in cancer progression has led to many discoveries of which mechanistic target of rapamycin (mTOR) is a key player. mTOR is a master regulator of cell growth control. mTOR is historically known to promote cell growth by enhancing the efficiency of protein translation. Research in the last decade has revealed that mTOR's role in promoting cell growth is much more multifaceted. While mTOR is necessary for normal human physiology, cancer cells take advantage of mTOR signalling to drive their neoplastic growth and progression. Oncogenic signal transduction through mTOR is a common occurrence in cancer, leading to metabolic transformation, enhanced proliferative drive and increased metastatic potential through neovascularisation. This review focuses on the downstream mTOR-regulated processes that are implicated in the "hallmarks" of cancer with focus on mTOR's involvement in proliferative signalling, metabolic reprogramming, angiogenesis and metastasis.

STAT3 and HIF1α Signaling Drives Oncogenic Cellular Phenotypes in Malignant Peripheral Nerve Sheath Tumors.

UNLABELLED: Therapeutic options are limited for neurofibromatosis type 1 (NF1)-associated malignant peripheral nerve sheath tumors (MPNST) and clinical trials using drug agents have so far been unsuccessful. This lack of clinical success is likely attributed to high levels of intratumoral molecular heterogeneity and variations in signal transduction within MPNSTs. To better explore the variance of malignant signaling properties within heterogeneous MPNSTs, four MPNST cell lines (ST8814, S462, S1844.1, and S1507.2) were used. The data demonstrate that small-molecule inhibition of the MET proto-oncogene and mTOR had variable outcome when preventing wound healing, cell migration, and invasion, with the S462 cells being highly resistant to both. Of interest, targeted inhibition of the STAT3 transcription factor suppressed wound healing, cell migration, invasion, and tumor formation in all four MPNST lines, which demonstrates that unlike MET and mTOR, STAT3 functions as a common driver of tumorigenesis in NF1-MPNSTs. Of clinical importance, STAT3 knockdown was sufficient to block the expression of hypoxia-inducible factor (HIF)1α, HIF2α, and VEGF-A in all four MPNST lines. Finally, the data demonstrate that wound healing, cell migration, invasion, and tumor formation through STAT3 are highly dependent on HIF signaling, where knockdown of HIF1α ablated these oncogenic facets of STAT3. IMPLICATIONS: This research reveals that aberrant STAT3 and HIF1a activity drives tumor progression in MPNSTs, indicating that inhibition of the STAT3/HIF1α/VEGF-A signaling axis is a viable treatment strategy.