The mitochondrial enzyme glutaminase (GLS) is frequently up-regulated during tumorigenesis and is being evaluated as a target for cancer therapy. GLS catalyzes the hydrolysis of glutamine to glutamate, which then supplies diverse metabolic pathways with carbon and/or nitrogen. Here, we report that SIRT5, a mitochondrial NAD+-dependent lysine deacylase, plays a key role in stabilizing GLS. In transformed cells, SIRT5 regulates glutamine metabolism by desuccinylating GLS and thereby protecting it from ubiquitin-mediated degradation. Moreover, we show that SIRT5 is up-regulated during cellular transformation and supports proliferation and tumorigenesis. Elevated SIRT5 expression in human breast tumors correlates with poor patient prognosis. These findings reveal a mechanism for increasing GLS expression in cancer cells and establish a role for SIRT5 in metabolic reprogramming and mammary tumorigenesis.

SUMMARY Efforts to target glutamine metabolism for cancer therapy have focused on the glutaminase isozyme GLS. The importance of the other isozyme, GLS2, in cancer has remained unclear, and it has been described as a tumor suppressor in some contexts. Here, we report that GLS2 is upregulated and essential in luminal-subtype breast tumors, which account for >70% of breast cancer incidence. We show that GLS2 expression is elevated by GATA3 in luminal-subtype cells but suppressed by promoter methylation in basal-subtype cells. Although luminal breast cancers resist GLS-selective inhibitors, we find that they can be targeted with a dual-GLS/GLS2 inhibitor. These results establish a critical role for GLS2 in mammary tumorigenesis and advance our understanding of how to target glutamine metabolism in cancer.

Protein kinases are essential mediators of cellular signal transduction and are often dysregulated in disease. Among these, protein tyrosine kinases (PTKs) have received specific interest due to their common roles in various diseases including cancer, and emerging observations indicating that PTK signalling pathways are susceptible to regulation by reactive oxygen species (ROS), which are also frequently implicated in disease pathology. While it is well recognized that ROS can impact on tyrosine kinase signalling by inhibiting tyrosine phosphatases, more recent studies highlight additional modes of redox-based regulation of tyrosine kinase signalling by direct redox modification of non-catalytic cysteines within tyrosine kinases or other protein components of this signalling pathway. In this review, we will present recent advancements with respect to redox-based mechanisms in regulating PTK signalling, with a specific focus on recent studies demonstrating direct redox regulation of Src-family kinases and epidermal growth factor receptor kinases. Importantly, redox-based modulation of tyrosine kinases may be relevant for many other kinases and has implications for current approaches to develop pharmacological inhibitors for these proteins.

Guanine nucleotide exchange factors (GEFs) activate Rho GTPases by catalyzing the exchange of bound GDP for GTP, thereby resulting in downstream effector recognition. Two metazoan families of GEFs have been described: Dbl-GEF family members that share conserved Dbl homology (DH) and Pleckstrin homology (PH) domains and the more recently described Dock180 family members that share little sequence homology with the Dbl family and are characterized by conserved Dock homology regions 1 and 2 (DHR-1 and -2). While extensive characterization of the Dbl family has been performed, less is known about how Dock180 family members act as GEFs, with only a single x-ray structure having recently been reported for the Dock9-Cdc42 complex. In order to learn more about the mechanisms used by the founding member of the family, Dock180, to act as a Rac-specific GEF, we set out to identify and characterize its limit functional GEF domain. A C-terminal portion of the DHR-2 domain, composed of approximately 300 residues (designated as Dock180 ), is shown to be necessary and sufficient for robust Rac-specific GEF activity both in vitro and in vivo. We further show that Dock180 binds to Rac in a manner distinct from Rac-GEFs of the Dbl family. Specifically, Ala 27 and Trp 56 of Rac appear to provide a bipartite binding site for the specific recognition of Dock180 , whereas, for Dbl family Rac-GEFs, Trp 56 of Rac is the sole primary determinant of GEF specificity. Based on our findings, we are able to define the core of Dock180 responsible for its Rac-GEF activity as well as highlight key recognition sites that distinguish different Dock180 family members and determine their corresponding GTPase specificities.Members of the Rho family of GTPases regulate a wide range of cellular activities including cell-cycle progression, gene transcription, cell migration, cell polarity, and vesicular trafficking through their abilities to bind to multiple downstream effectors (1-4). Rho GTPases switch between two states, the GDP-bound inactive state and the GTP-bound active state. Tight regulation of Rho GTPases and their nucleotide-bound state is important for mediating their different cellular functions (5). Three main classes of regulatory proteins for Rho GTPases have been identified and characterized. Guanine nucleotide exchange factors (GEFs) include ~70 mammalian/human proteins that promote the exchange of GDP for GTP on Rho GTPases. GTPase-activating proteins (GAPs) catalyze the hydrolysis of the bound GTP to GDP, and Rho GDP-dissociation inhibitors (GDIs) slow nucleotide exchange while serving to sequester Rho GTPases in the cytoplasm (6-8).Two families of GEFs have been discovered for Rho GTPases, which we refer to here as the Dbl (9) and Dock180 superfamilies (10-12). The Dbl family members all possess two † This work was supported by National Institutes of Health R01 Grants GM40654 and GM47458.* To whom correspondence should be addressed: Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Itha...

Over-expression of the receptor tyrosine kinase HER2/ErbB2 (ERBB2) has been linked to a poor prognosis for breast cancer patients; thus, its activity is a central target for cancer therapy. Likewise, over-expression of heregulin (HRG/NRG1), a growth factor responsible for ErbB2 activation, has also been shown to be a driver of breast cancer progression. Although ErbB2 inhibitors offer a major advancement in the treatment of ErbB2-dependent breast cancers, patients are highly susceptible to developing clinical resistance to these drugs. Therefore, a detailed understanding of the molecular mechanism that underlies HRG/ErbB2-induced tumorigenesis is essential for the development of effective therapeutic strategies for this subset of breast cancer patients. Here, it was demonstrated that HRG promoted anchorage-independent breast cancer cell growth more potently than EGF, and that the HRG-dependent activation of PI3K and mTORC1 are necessary events for cell transformation. Functional evaluation of two distinct mTOR inhibitors, rapamycin and INK-128, on HRG-dependent signaling activities, uncovered a necessary role for mTORC2 in the regulation of the AKT/TSC2/mTORC1 axis by impacting the phosphorylation of AKT at the PDK1-dependent site (T308) as well as at the mTORC2-dependent site (S473). The elimination of Rictor, a critical component of mTORC2, is detrimental to both the activation of mTORC1 and HRG-mediated cellular transformation. Similar results were obtained in multiple breast cancer model systems, highlighting an important role for mTORC2 in HRG/ErbB2-dependent breast cancer. Implications These findings suggest the potential benefits of targeting mTORC2 in HRG/ErbB2-induced breast cancer.