Pierdomenico Ruggeri

Thioredoxin stimulates MMP‐9 expression, de‐regulates the MMP‐9/TIMP‐1 equilibrium and promotes MMP‐9 dependent invasion in human MDA‐MB‐231 breast cancer cells

Increased expression of thioredoxin (Trx)‐1 and matrix metalloproteinase (MMP)‐9 associates with malignant breast cancer progression. Here, we describe a functional relationship between Trx‐1 and MMP‐9 in promoting MDA‐MB‐231 breast cancer cell invasive behaviour. Trx‐1 overexpression stimulated MMP‐9 expression, de‐regulated the MMP‐9/TIMP‐1 equilibrium and augmented MMP‐9 involvement in a more invasive phenotype. Trx‐1 augmented MMP‐9 transcription through NF‐κB, AP‐1 and SP1 elements; stimulated p50/p65 NF‐κB activity and recruitment to the MMP‐9 promoter; and facilitated MMP‐9 promoter‐accessibility to NF‐κB by preventing HDAC recruitment and maintaining MMP‐9 promoter histone acetylation. Our data provide a functional basis for Trx‐1 and MMP‐9 association in malignant breast cancer and identify Trx‐1 and NF‐κB as potentially druggable targets for reducing MMP‐9 involvement in malignant behaviour.

Constitutive autotaxin transcription by Nmyc-amplified and non-amplified neuroblastoma cells is regulated by a novel AP-1 and SP-mediated mechanism and abrogated by curcumin

The motility, angiogenesis and metastasis‐stimulating factor Autotaxin (Atx), over expressed by human neuroblastomas (NB), is constitutively expressed by human Nmyc‐amplified SK‐N‐BE and non‐Nmyc‐amplified SH‐SY5Y NB cells. Here, we characterise a novel Atx transcriptional mechanism, utilised by both cell lines, that is restricted to the first 285 bp of the Atx promoter and involves AP‐1 and SP transcription factors, acting through a CRE/AP‐1‐like element at position −142 to −149 and a GAbox at position −227 to −235 relative to the Atx translational start site. This novel transcriptional mechanism can be inhibited by internally initiated SP‐3 and the natural phenol curcumin.

The TrkAIII Oncoprotein Inhibits Mitochondrial Free Radical ROS-Induced Death of SH-SY5Y Neuroblastoma Cells by Augmenting SOD2 Expression and Activity at the Mitochondria, within the Context of a Tumour Stem Cell-like Phenotype

The developmental and stress-regulated alternative TrkAIII splice variant of the NGF receptor TrkA is expressed by advanced stage human neuroblastomas (NBs), correlates with worse outcome in high TrkA expressing unfavourable tumours and exhibits oncogenic activity in NB models. In the present study, we report that constitutive TrkAIII expression in human SH-SY5Y NB cells inhibits Rotenone, Paraquat and LY83583-induced mitochondrial free radical reactive oxygen species (ROS)-mediated death by stimulating SOD2 expression, increasing mitochondrial SOD2 activity and attenuating mitochondrial free radical ROS production, in association with increased mitochondrial capacity to produce H2O2, within the context of a more tumour stem cell-like phenotype. This effect can be reversed by the specific TrkA tyrosine kinase inhibitor GW441756, by the multi-kinase TrkA inhibitors K252a, CEP-701 and Gö6976, which inhibit SOD2 expression, and by siRNA knockdown of SOD2 expression, which restores the sensitivity of TrkAIII expressing SH-SY5Y cells to Rotenone, Paraquat and LY83583-induced mitochondrial free radical ROS production and ROS-mediated death. The data implicate the novel TrkAIII/SOD2 axis in promoting NB resistance to mitochondrial free radical-mediated death and staminality, and suggest that the combined use of TrkAIII and/or SOD2 inhibitors together with agents that induce mitochondrial free radical ROS-mediated death could provide a therapeutic advantage that may also target the stem cell niche in high TrkA expressing unfavourable NB.

Alendronate promotes plasmin-mediated MMP-9 inactivation by exposing cryptic plasmin degradation sites within the MMP-9 catalytic domain

Irreversible MMP‐9 inhibition is considered a significant therapeutic goal in inflammatory, vascular and tumour pathology. We report that divalent cation chelators Alendronate and EDTA not only directly inhibited MMP‐9 but also promoted irreversible plasmin‐mediated MMP‐9 inactivation by exposing cryptic plasmin‐degradation sites within the MMP‐9 catalytic‐domain and producing an inhibitory hemopexin–domain fragment. This effect was also observed using MDA‐MB‐231 breast cancer cells, which activated exogenous plasminogen to degrade endogenous proMMP‐9 in the presence of Alendronate or EDTA. Degradation‐mediated inactivation of proMMP‐9 occurred in the absence of transient activation, attesting to the incapacity of plasmin to directly activate proMMP‐9 and direct MMP‐9 inhibition by Alendronate and EDTA. Our study provides a novel rational for therapeutic Alendronate use in MMP‐9‐dependent pathology characterised by plasminogen activation.

Retrograde TrkAIII transport from ERGIC to ER: a re-localisation mechanism for oncogenic activity

In human SH-SY5Y neuroblastoma (NB) cells, nascent immature N-glycosylated 110kDa TrkA moves rapidly from the endoplasmic reticulum (ER) to the Golgi Network (GN), where it matures into the 140kDa receptor prior to being transported to the cell surface, creating GN and cell surface pools of inactive receptor maintained below the spontaneous activation threshold by a full compliment of inhibitory domains and endogenous PTPases. In contrast, the oncogenic alternative TrkAIII splice variant is not expressed at the cell surface but re-localises to intracellular membranes, within which it exhibits spontaneous ERGIC/COPI-associated activation and oncogenic Akt signalling. In this study, we characterise the mechanism responsible for TrkAIII re-localisation. Spontaneous TrkAIII activation, facilitated by D4 IG-like domain and N-glycosylation site omission, increases spontaneous activation potential by altering intracellular trafficking, inhibiting cell surface expression and eliminating an important inhibitory domain. TrkAIII, spontaneously activated within the permissive ERGIC/COPI compartment, rather than moving in an anterograde direction to the GN exhibits retrograde transport back to the ER, where it is inactivated. This sets-up self-perpetuating TrkAIII re-cycling between the ERGIC and ER, that ensures continual accumulation above the spontaneous activation threshold of the ERGIC/COPI compartment. This is reversed by TrkA tyrosine kinase inhibitors, which promote anterograde transport of inactivated TrkAIII to the GN, resulting in GN-associated TrkAIII maturation to a 120kDa species that is degraded at the proteasome.

TrkAIII Promotes Microtubule Nucleation and Assembly at the Centrosome in SH-SY5Y Neuroblastoma Cells, Contributing to an Undifferentiated Anaplastic Phenotype

The alternative TrkAIII splice variant is expressed by advanced stage human neuroblastomas (NBs) and exhibits oncogenic activity in NB models. In the present study, employing stable transfected cell lines and assays of indirect immunofluorescence, immunoprecipitation, Western blotting, microtubule regrowth, tubulin kinase, and tubulin polymerisation, we report that TrkAIII binds α-tubulin and promotes MT nucleation and assembly at the centrosome. This effect depends upon spontaneous TrkAIII activity, TrkAIII localisation to the centrosome and pericentrosomal area, and the capacity of TrkAIII to bind, phosphorylate, and polymerise tubulin. We propose that this novel role for TrkAIII contributes to MT involvement in the promotion and maintenance of an undifferentiated anaplastic NB cell morphology by restricting and augmenting MT nucleation and assembly at the centrosomal MTOC.

Neurotrophin and Neurotrophin Receptor Involvement in Human Neuroblastoma

Neuroblastoma (NB) is an embryonic tumour that originates from cells of the neural crest (NC) arrested in their differentiation at different stages along the sympatho-adrenal lineage and, less frequently, from precursors of sensory neurons [1, 2]. As a consequence, NB can occur throughout the sympathetic chain from thoracic, abdominal and pelvic sites to the adrenal medulla, which accounts for the majority of NBs. Consistent with this, NBs exhibit a high degree of genetic heterogeneity and biological variability, including differences in catechola‐ mine expression, according to their differentiation state along the sympathoadrenal lineage, with a small number of primitive midline and spinal NBs that do not secrete catecholamines considered to be of dorsal root sensory origin [1, 2].

Alternative TrkA Splicing and Neuroblastoma

Neuroblastoma is one of the most frequent solid paediatric tumours of the nervous system, accounting for up to 10% of all paediatric tumours. The majority of NBs originate from a sympathoadrenal cell lineage of neural crest origin during sympathetic nervous system development, and represent a heterogeneous group of tumours that exhibit a high degree of genetic and biological variability, including not infrequent spontaneous regression or differentiation to ganglioneuroma (Evans, 2004; Nakagawara, 2004). A large percentage of NB patients present with stage 4 disease characterised by dissemination primarily to bone; bone marrow; lymph node; liver and skin sites, with metastatic bone disease carrying automatic stage 4 diagnosis and the poorest prognosis. A subset of stage 4 NBs that disseminate primarily to liver skin and bone marrow sites exhibit frequent spontaneous regression and are classified as stage 4S. Genetic alterations that associated with aggressive NB include: amplification of the proto-oncogenic transcription factor N-myc in up to 20% of all NBs and up to 40% of aggressive NB; gain of chromosome 17 and loss of distal material from the chromosomes 1p32-pter (minimal common region 1p36.2); 14p23-qter; 11q23 and 18, regions likely to contain oncosuppressors (Evans, 2004; Nakagawara, 2004; Jiang et al., 2011; Takita et al., 2000). Despite general improvements in therapy, the age of onset plus high frequency of post-therapeutic relapse have meant that survival rates in patients with NB remain poor, highlighting the need for a greater understanding of the molecular mechanisms involved in this tumor type and the translation of this information into novel therapies. Receptor tyrosine kinases (RTKs) regulate cellular growth, differentiation and survival during development. In general, RTK function depends upon appropriate ligand binding, with inappropriate activation and temporary duration of activation regulated by molecular domain, glycosylation status; protein chaperones; phosphorylation status and associated protein tyrosine phosphatases. The deregulation of RTK function is involved in tumour pathology, with over 30 RTKs associated with different malignancies, and is associated with

TrkAIII signals endoplasmic reticulum stress to the mitochondria in neuroblastoma cells, resulting in glycolytic metabolic adaptation

Alternative TrkAIII splicing characterises advanced stage metastatic disease and post-therapeutic relapse in neuroblastoma (NB), and in NB models TrkAIII exhibits oncogenic activity. In this study, we report a novel role for TrkAIII in signaling ER stress to the mitochondria in SH-SY5Y NB cells that results in glycolytic metabolic adaptation. The ER stress-inducing agents DTT, A23187 and thapsigargin activated the ER stress-response in control pcDNA SH-SY5Y and TrkAIII expressing SH-SY5Y cells and in TrkAIII SH-SY5Y cells increased TrkAIII targeting to mitochondria and internalisation into inner-mitochondrial membranes. Within inner-mitochondrial membranes, TrkAIII was subjected to Omi/HtrA2-dependent cleavage to tyrosine phosphorylated 45–48kDa carboxyl terminal active fragments, localised predominantly in tyrosine kinase-domain mitochondrial matrix orientation. This stress-induced activation of mitochondrial TrkAIII was associated with increased ROS production, prevented by the ROS scavenger Resveratrol and underpinned by changes in Ca2+ movement, implicating ROS/Ca2+ interplay in overcoming the mitochondrial TrkAIII activation threshold. Stress-induced, cleavage-activation of mitochondrial TrkAIII resulted in mitochondrial PDHK1 tyrosine phosphorylation, leading to glycolytic metabolic adaptation. This novel mitochondrial role for TrkAIII provides a potential self-perpetuating, drug reversible way through which tumour microenvironmental stress may maintain the metastasis promoting “Warburg effect” in TrkAIII expressing NBs.

TRAIL induces pro-apoptotic crosstalk between the TRAIL-receptor signaling pathway and TrkAIII in SH-SY5Y cells, unveiling a potential therapeutic “Achilles heel” for the TrkAIII oncoprotein in neuroblastoma

TrkAIII expression in neuroblastoma (NB) associates with advanced stage disease, worse prognosis, post therapeutic relapse, and in NB models TrkAIII exhibits oncogenic activity and promotes chemotherapeutic-resistance. Here, we report a potential therapeutic “Achilles heel” for the TrkAIII oncoprotein in a SH-SY5Y NB model that is characterised by one-way TRAIL-induced, pro-apoptotic crosstalk between the TRAIL receptor signaling pathway and TrkAIII that results in the delayed induction of apoptosis. In TrkAIII SH-SY5Y cells, blocked in the intrinsic apoptosis pathway by elevated constitutive Bcl-2, Bcl-xL and Mcl-1 expression, TRAIL induced delayed caspase-dependent apoptosis via the extrinsic pathway and completely abrogated tumourigenic capacity in vitro. This effect was initiated by TRAIL-induced SHP-dependent c-Src activation, the induction of TrkAIII/SHP-1/c-Src complexing leading to SHP-mediated TrkAIII de-phosphorylation, subsequent induction of complexing between de-phosphorylated TrkAIII and cFLIP associated with a time-dependent increase the caspase-8 to cFLIP ratio at activated death receptors, resulting in delayed caspase cleavage and caspase-dependent apoptosis. We also confirm rate-limiting roles for c-FLIP and Mcl-1 in regulating the sensitivity of TrkAIII SH-SY5Y cells to TRAIL-induced apoptosis via the extrinsic and intrinsic pathways, respectively. Our study unveils a novel mechanism for the TRAIL-induced apoptosis of TrkAIII expressing NB cells that depends upon SHP/Src-mediated crosstalk between the TRAIL-receptor signaling pathway and TrkAIII, and supports a novel potential pro-apoptotic therapeutic use for TRAIL in TrkAIII expressing NB.