Alessio Giubellino

Grb2 signaling in cell motility and cancer

Background: Metastasis is the primary cause of death in most human cancers, and understanding the molecular mechanisms underpinning this multistep process is fundamental to identifying novel molecular targets and developing more effective therapies. Objective/methods: Here we review the role of growth factor receptor-bound protein 2 (Grb2) in cancer and specifically in metastasis-related processes, and summarize the development of anticancer therapeutics selectively targeting this adapter protein. Results/conclusion: Grb2 is a key molecule in intracellular signal transduction, linking activated cell surface receptors to downstream targets by binding to specific phosphotyrosine-containing and proline-rich sequence motifs. Grb2 signaling is critical for cell cycle progression and actin-based cell motility, and, consequently, more complex processes such as epithelial morphogenesis, angiogenesis and vasculogenesis. These functions make Grb2 a therapeutic target for strategies designed to prevent the spread of solid tumors through local invasion and metastasis.

c-Met ectodomain shedding rate correlates with malignant potential.

Purpose: Many proteins are proteolytically released from the cell surface by a process known as ectodomain shedding. Shedding occurs under normal physiologic conditions and can be increased in certain pathologies. Among the many receptors for which ectodomain shedding has been shown is c-Met, the hepatocyte growth factor (HGF) receptor tyrosine kinase. HGF stimulates mitogenesis, motogenesis, and morphogenesis in a variety of cellular targets during development, homeostasis, and tissue regeneration. Inappropriate HGF signaling resulting in unregulated cell proliferation, motility, and invasion occurs in several human malignancies. This can occur through paracrine signaling, autocrine loop formation, receptor mutation, gene amplification, or gene rearrangement, accompanied frequently with overexpression of ligand and/or receptor proteins. We hypothesized that c-Met overexpression in cancer might result in increased ectodomain shedding, and that its measure could be a useful biomarker of tumor progression. Experimental Design: We developed a sensitive electrochemiluminescent immunoassay to quantitate c-Met protein in cell lysates, culture supernatants, and biological samples. Results: A survey of cultured cell models of oncogenic transformation revealed significant direct correlations (P < 0.001, t test or ANOVA) between malignant potential and the rate of c-Met ectodomain shedding that was independent of steady-state receptor expression level. Moreover, weekly plasma and urine samples from mice harboring s.c. human tumor xenografts (n = 4 per group) displayed soluble human c-Met levels that were measurable before tumors became palpable and that correlated directly with tumor volume (R2 > 0.92, linear regression). Conclusions: For a variety of human cancers, c-Met ectodomain shedding may provide a reliable and practical indicator of malignant potential and overall tumor burden.

Hereditary papillary renal carcinoma type I

Germline missense mutations in the tyrosine kinase domain of the hepatocyte growth factor/scatter factor (HGF/SF) receptor, c-Met, are thought to be responsible for hereditary papillary renal carcinoma (HPRC) type 1, a form of human kidney cancer. In addition to extensive linkage analysis of HPRC families localizing the HPRC type 1 gene within chromosome 7, the demonstration that individual c-Met mutations reconstituted in cultured cells display enhanced and dysregulated kinase activity, and confer cell transformation and tumorigenicity in mice, solidifies this conclusion. Our prior knowledge of HGF/SF biology and c-Met signaling enabled rapid progress in unraveling the molecular pathogenesis of HPRC type 1, and in laying the framework for the development of novel therapeutics for the treatment of this cancer. At the same time, the study of HPRC type 1 has refined our appreciation of the oncogenic potential of c-Met signaling, and challenges our current understanding of HGF/SF and c-Met function in health and disease.

Targeting the Met signaling pathway in renal cancer

Renal cell carcinoma (RCC), the most common form of kidney cancer, accounts for 3% of all adult malignancies and its incidence has significantly increased over the last 20 years. RCC claims 13,000 lives annually in the USA and more than 100,000 worldwide. A better understanding of the molecular basis of RCC has facilitated the development of novel and more selective therapeutic approaches. An important role in RCC oncogenesis is played by the receptor for HGF, Met, which has attracted considerable attention, more recently as a molecular target for cancer therapy, and several drugs selectively targeting this pathway are now in clinical trials. This review will focus on efforts to understand the role of the Met signaling pathway in renal cancer and how this has contributed to the development of potent and selective drug candidates.

Pituitary Adenoma With Paraganglioma/Pheochromocytoma (3PAs) and Succinate Dehydrogenase Defects in Humans and Mice

CONTEXT Germline mutations in genes coding succinate dehydrogenase (SDH) subunits A, B, C, and D have been identified in familial paragangliomas (PGLs)/pheochromocytomas (PHEOs) and other tumors. We described a GH-secreting pituitary adenoma (PA) caused by SDHD mutation in a patient with familial PGLs. Additional patients with PAs and SDHx defects have since been reported. DESIGN We studied 168 patients with unselected sporadic PA and with the association of PAs, PGLs, and/or pheochromocytomas, a condition we named the 3P association (3PAs) for SDHx germline mutations. We also studied the pituitary gland and hormonal profile of Sdhb(+/-) mice and their wild-type littermates at different ages. RESULTS No SDHx mutations were detected among sporadic PA, whereas three of four familial cases were positive for a mutation (75%). Most of the SDHx-deficient PAs were either prolactinomas or somatotropinomas. Pituitaries of Sdhb(+/-) mice older than 12 months had an increased number mainly of prolactin-secreting cells and several ultrastructural abnormalities such as intranuclear inclusions, altered chromatin nuclear pattern, and abnormal mitochondria. Igf-1 levels of mutant mice tended to be higher across age groups, whereas Prl and Gh levels varied according to age and sex. CONCLUSION The present study confirms the existence of a new association that we termed 3PAs. It is due mostly to germline SDHx defects, although sporadic cases of 3PAs without SDHx defects also exist. Using Sdhb(+/-) mice, we provide evidence that pituitary hyperplasia in SDHx-deficient cells may be the initial abnormality in the cascade of events leading to PA formation.

Microarray analysis sheds light on the dedifferentiating role of agouti signal protein in murine melanocytes via the Mc1r

The melanocortin-1 receptor (MC1R) is a key regulator of pigmentation in mammals and is tightly linked to an increased risk of skin cancers, including melanoma, in humans. Physiologically activated by α-melanocyte stimulating hormone (αMSH), MC1R function can be antagonized by a secreted factor, agouti signal protein (ASP), which is responsible for the lighter phenotypes in mammals (including humans), and is also associated with increased risk of skin cancer. It is therefore of great interest to characterize the molecular effects elicited by those MC1R ligands. In this study, we determined the gene expression profiles of murine melan-a melanocytes treated with ASP or αMSH over a 4-day time course using genome-wide oligonucleotide microarrays. As expected, there were significant reductions in expression of numerous melanogenic proteins elicited by ASP, which correlates with its inhibition of pigmentation. ASP also unexpectedly modulated the expression of genes involved in various other cellular pathways, including glutathione synthesis and redox metabolism. Many genes up-regulated by ASP are involved in morphogenesis (especially in nervous system development), cell adhesion, and extracellular matrix-receptor interactions. Concomitantly, ASP enhanced the migratory potential and the invasiveness of melanocytic cells in vitro. These results demonstrate the role of ASP in the dedifferentiation of melanocytes, identify pigment-related genes targeted by ASP and by αMSH, and provide insights into the pleiotropic molecular effects of MC1R signaling that may function during development and may affect skin cancer risk.

Directed Discovery of Agents Targeting the Met Tyrosine Kinase Domain by Virtual Screening

Hepatocyte growth factor (HGF) is an important regulator of normal development and homeostasis, and dysregulated signaling through the HGF receptor, Met, contributes to tumorigenesis, tumor progression, and metastasis in numerous human malignancies. The development of selective small-molecule inhibitors of oncogenic tyrosine kinases (TK) has led to well-tolerated, targeted therapies for a growing number of cancer types. To identify selective Met TK inhibitors, we used a high-throughput virtual screen of the 13.5 million compound ChemNavigator database to find compounds most likely to bind to the Met ATP binding site and to form several critical interactions with binding site residues predicted to stabilize the kinase domain in its inactive conformation. Subsequent biological screening of 70 in silico hit structures using cell-free and intact cell assays identified three active compounds with micromolar IC(50) values. The predicted binding modes and target selectivity of these compounds are discussed and compared to other known Met TK inhibitors.

Inhibition of Tumor Metastasis by a Growth Factor Receptor Bound Protein 2 Src Homology 2 Domain–Binding Antagonist

Metastasis, the primary cause of death in most forms of cancer, is a multistep process whereby cells from the primary tumor spread systemically and colonize distant new sites. Blocking critical steps in this process could potentially inhibit tumor metastasis and dramatically improve cancer survival rates; however, our understanding of metastasis at the molecular level is still rudimentary. Growth factor receptor binding protein 2 (Grb2) is a widely expressed adapter protein with roles in epithelial cell growth and morphogenesis, as well as angiogenesis, making it a logical target for anticancer drug development. We have previously shown that a potent antagonist of Grb2 Src homology-2 domain-binding, C90, blocks growth factor-driven cell motility in vitro and angiogenesis in vivo. We now report that C90 inhibits metastasis in vivo in two aggressive tumor models, without affecting primary tumor growth rate. These results support the potential efficacy of this compound in reducing the metastatic spread of primary solid tumors and establish a critical role for Grb2 Src homology-2 domain-mediated interactions in this process.

Combined inhibition of mTORC1 and mTORC2 signaling pathways is a promising therapeutic option in inhibiting pheochromocytoma tumor growth: in vitro and in vivo studies in female athymic nude mice.

Several lines of evidence, including the recent discovery of novel susceptibility genes, point out an important role for the mammalian target of rapamycin (mTOR) signaling pathway in the development of pheochromocytoma. Analyzing a set of pheochromocytomas from patients with different genetic backgrounds, we observed and confirmed a significant overexpression of key mTOR complex (mTORC) signaling mediators. Using selective ATP-competitive inhibitors targeting both mTORC1 and mTORC2, we significantly arrested the in vitro cell proliferation and blocked migration of pheochromocytoma cells as a result of the pharmacological suppression of the Akt/mTOR signaling pathway. Moreover, AZD8055, a selective ATP-competitive dual mTORC1/2 small molecular inhibitor, significantly reduced the tumor burden in a model of metastatic pheochromocytoma using female athymic nude mice. This study suggests that targeting both mTORC1 and mTORC2 is a potentially rewarding strategy and supports the application of selective inhibitors in combinatorial drug regimens for metastatic pheochromocytoma.

NF-κB inhibition significantly upregulates the norepinephrine transporter system, causes apoptosis in pheochromocytoma cell lines and prevents metastasis in an animal model

Pheochromocytomas (PHEOs) and paragangliomas (PGLs) are specific types of neuroendocrine tumors that originate in the adrenal medulla or sympathetic/parasympathetic paraganglia, respectively. Although these tumors are intensively studied, a very effective treatment for metastatic PHEO or PGL has not yet been established. Preclinical evaluations of novel therapies for these tumors are very much required. Therefore, in this study we tested the effect of triptolide (TTL), a potent nuclear factor‐kappaB (NF‐κB) inhibitor, on the cell membrane norepinephrine transporter (NET) system, considered to be the gatekeeper for the radiotherapeutic agent 131I‐metaiodobenzylguanidine (131I‐MIBG). We measured changes in the mRNA and protein levels of NET and correlated them with proapoptotic factors and metastasis inhibition. The study was performed on three different stable PHEO cell lines. We found that blocking NF‐κB with TTL or capsaicin increased both NET mRNA and protein levels. Involvement of NF‐κB in the upregulation of NET was verified by mRNA silencing of this site and also by using NF‐κB antipeptide. Moreover, in vivo treatment with TTL significantly reduced metastatic burden in an animal model of metastatic PHEO. The present study for the first time shows how NF‐κB inhibitors could be successfully used in the treatment of metastatic PHEO/PGL by a significant upregulation of NET to increase the efficacy of 131I‐MIBG and by the induction of apoptosis.