Faculty Bibliography

Genomic analyses are yielding a host of new information on the multiple genetic abnormalities associated with specific types of cancer. A comprehensive description of cancer-associated genetic abnormalities can improve our ability to classify tumors into clinically relevant subgroups and, on occasion, identify mutant genes that drive the cancer phenotype ("drivers"). More often, though, the functional significance of cancer-associated mutations is difficult to discern. Genome-wide pooled short hairpin RNA (shRNA) screens enable global identification of the genes essential for cancer cell survival and proliferation, providing a "functional genomic" map of human cancer to complement genomic studies. Using a lentiviral shRNA library targeting ~16,000 genes and a newly developed, dynamic scoring approach, we identified essential gene profiles in 72 breast, pancreatic, and ovarian cancer cell lines. Integrating our results with current and future genomic data should facilitate the systematic identification of drivers, unanticipated synthetic lethal relationships, and functional vulnerabilities of these tumor types. SIGNIFICANCE: This study presents a resource of genome-scale, pooled shRNA screens for 72 breast, pancreatic, and ovarian cancer cell lines that will serve as a functional complement to genomics data, facilitate construction of essential gene profiles, help uncover synthetic lethal relationships, and identify uncharacterized genetic vulnerabilities in these tumor types. SIGNIFICANCE: This study presents a resource of genome-scale, pooled shRNA screens for 72 breast, pancreatic, and ovarian cancer cell lines that will serve as a functional complement to genomics data, facilitate construction of essential gene profiles, help uncover synthetic lethal relationships, and identify uncharacterized genetic vulnerabilities in these tumor types.

AIMS/HYPOTHESIS: Insulin activates insulin receptor protein tyrosine kinase and downstream phosphatidylinositol-3-kinase (PI3K)/Akt signalling in muscle to promote glucose uptake. The insulin receptor can serve as a substrate for the protein tyrosine phosphatase (PTP) 1B and T cell protein tyrosine phosphatase (TCPTP), which share a striking 74% sequence identity in their catalytic domains. PTP1B is a validated therapeutic target for the alleviation of insulin resistance in type 2 diabetes. PTP1B dephosphorylates the insulin receptor in liver and muscle to regulate glucose homeostasis, whereas TCPTP regulates insulin receptor signalling and gluconeogenesis in the liver. In this study we assessed for the first time the role of TCPTP in the regulation of insulin receptor signalling in muscle. METHODS: We generated muscle-specific TCPTP-deficient (Mck-Cre;Ptpn2(lox/lox)) mice (Mck, also known as Ckm) and assessed the impact on glucose homeostasis and muscle insulin receptor signalling in chow-fed versus high-fat-fed mice. RESULTS: Blood glucose and insulin levels, insulin and glucose tolerance, and insulin-induced muscle insulin receptor activation and downstream PI3K/Akt signalling remained unaltered in chow-fed Mck-Cre;Ptpn2(lox/lox) versus Ptpn2(lox/lox) mice. In addition, body weight, adiposity, energy expenditure, insulin sensitivity and glucose homeostasis were not altered in high-fat-fed Mck-Cre;Ptpn2(lox/lox) versus Ptpn2(lox/lox) mice. CONCLUSIONS/INTERPRETATION: These results indicate that TCPTP deficiency in muscle has no effect on insulin signalling and glucose homeostasis, and does not prevent high-fat diet-induced insulin resistance. Thus, despite their high degree of sequence identity, PTP1B and TCPTP contribute differentially to insulin receptor regulation in muscle. Our results are consistent with the notion that these two highly related PTPs make distinct contributions to insulin receptor regulation in different tissues.

Protein tyrosine phosphatase 1B (PTP1B), a key negative regulator of leptin and insulin signaling, is positively correlated with adiposity and contributes to insulin resistance. Global PTP1B deletion improves diet-induced obesity and glucose homeostasis via enhanced leptin signaling in the brain and increased insulin signaling in liver and muscle. However, the role of PTP1B in adipocytes is unclear, with studies demonstrating beneficial, detrimental or no effect(s) of adipose-PTP1B-deficiency on body mass and insulin resistance. To definitively establish the role of adipocyte-PTP1B in body mass regulation and glucose homeostasis, adipocyte-specific-PTP1B knockout mice (adip-crePTP1B(-/-)) were generated using the adiponectin-promoter to drive Cre-recombinase expression. Chow-fed adip-crePTP1B(-/-) mice display enlarged adipocytes, despite having similar body weight/adiposity and glucose homeostasis compared to controls. High-fat diet (HFD)-fed adip-crePTP1B(-/-) mice display no differences in body weight/adiposity but exhibit larger adipocytes, increased circulating glucose and leptin levels, reduced leptin sensitivity and increased basal lipogenesis compared to controls. This is associated with decreased insulin receptor (IR) and Akt/PKB phosphorylation, increased lipogenic gene expression and increased hypoxia-induced factor-1-alpha (Hif-1alpha) expression. Adipocyte-specific PTP1B deletion does not beneficially manipulate signaling pathways regulating glucose homeostasis, lipid metabolism or adipokine secretion in adipocytes. Moreover, PTP1B does not appear to be the major negative regulator of the IR in adipocytes.

Protein-tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed PTP that is anchored to the endoplasmic reticulum (ER). PTP1B dephosphorylates activated receptor tyrosine kinases after endocytosis, as they transit past the ER. However, PTP1B also can access some plasma membrane (PM)-bound substrates at points of cell-cell contact. To explore how PTP1B interacts with such substrates, we utilized quantitative cellular imaging approaches and mathematical modeling of protein mobility. We find that the ER network comes in close proximity to the PM at apparently specialized regions of cell-cell contact, enabling PTP1B to engage substrate(s) at these sites. Studies using PTP1B mutants show that the ER anchor plays an important role in restricting its interactions with PM substrates mainly to regions of cell-cell contact. In addition, treatment with PTP1B inhibitor leads to increased tyrosine phosphorylation of EphA2, a PTP1B substrate, specifically at regions of cell-cell contact. Collectively, our results identify PM-proximal sub-regions of the ER as important sites of cellular signaling regulation by PTP1B.

In obesity, anorectic responses to leptin are diminished, giving rise to the concept of "leptin resistance." Increased expression of protein tyrosine phosphatase 1B (PTP1B) has been associated with the attenuation of leptin signaling and development of cellular leptin resistance. Here we report that hypothalamic levels of the tyrosine phosphatase TCPTP are also elevated in obesity to attenuate the leptin response. We show that mice that lack TCPTP in neuronal cells have enhanced leptin sensitivity and are resistant to high-fat-diet-induced weight gain and the development of leptin resistance. Also, intracerebroventricular administration of a TCPTP inhibitor enhances leptin signaling and responses in mice. Moreover, the combined deletion of TCPTP and PTP1B in neuronal cells has additive effects in the prevention of diet-induced obesity. Our results identify TCPTP as a critical negative regulator of hypothalamic leptin signaling and causally link elevated TCPTP to the development of cellular leptin resistance in obesity.

Reactive oxygen species (ROS) regulate growth factor receptor signalling at least in part by inhibiting oxidation-sensitive phosphatases. An emerging concept is that ROS act locally to affect signal transduction in different subcellular compartments and that ROS levels are regulated by antioxidant proteins at the same local level. Here, we show that the ER-resident antioxidant peroxiredoxin 4 (Prdx4) interacts with the cytoplasmic domain of the granulocyte colony-stimulating factor receptor (G-CSFR). This interaction occurs when the activated G-CSFR resides in early endosomes. Prdx4 inhibits G-CSF-induced signalling and proliferation in myeloid progenitors, depending on its redox-active cysteine core. Protein tyrosine phosphatase 1b (Ptp1b) appears to be a major downstream effector controlling these responses. Conversely, Ptp1b might keep Prdx4 active by reducing its phosphorylation. These findings unveil a new signal transduction regulatory circuitry involving redox-controlled processes in the ER and activated cytokine receptors in endosomes.

The biological response to tumor necrosis factor (TNF) involves activation of MAP kinases. Here we report a mechanism of MAP kinase activation by TNF that is mediated by the Rho GTPase family members Rac/Cdc42. This signaling pathway requires Src-dependent activation of the guanosine nucleotide exchange factor Vav, activation of Rac/Cdc42, and the engagement of the Rac/Cdc42 interaction site (CRIB motif) on mixed-lineage protein kinases (MLKs). We show that this pathway is essential for full MAP kinase activation during the response to TNF. Moreover, this MLK pathway contributes to inflammation in vivo.

Protein-tyrosine phosphatase 1B (PTP1B), a well-established metabolic regulator, plays an important role in breast cancer. Using whole-body PTP1B knockout mice, recent studies have shown that PTP1B ablation delays HER2/Neu-induced mammary cancer. Whether PTP1B plays a cell-autonomous or a noncell-autonomous role in HER2/Neu-evoked tumorigenesis and whether it is involved in tumor maintenance was unknown. We generated mice expressing HER2/Neu and lacking PTP1B specifically in the mammary epithelium. We found that mammary-specific deletion of PTP1B delays the onset of HER2/Neu-evoked mammary tumors, establishing a cell autonomous role for PTP1B in such neoplasms. We also deleted PTP1B in established mouse mammary tumors or depleted PTP1B in human breast cancer cell lines grown as xenografts. PTP1B inhibition did not affect tumor growth in either model showing that neither epithelial nor stromal PTP1B is necessary for tumor maintenance. Taken together, our data show that despite the PTP1B contribution to tumor onset, it is not essential for tumor maintenance. This suggests that PTP1B inhibition could be effective in breast tumor prevention.

Protein-tyrosine phosphatases (PTPs), along with protein-tyrosine kinases, play key roles in cellular signaling. All Class I PTPs contain an essential active site cysteinyl residue, which executes a nucleophilic attack on substrate phosphotyrosyl residues. The high reactivity of the catalytic cysteine also predisposes PTPs to oxidation by reactive oxygen species, such as H(2)O(2). Reversible PTP oxidation is emerging as an important cellular regulatory mechanism and might contribute to diseases such as cancer. We exploited these unique features of PTP enzymology to develop proteomic methods, broadly applicable to cell and tissue samples, that enable the comprehensive identification and quantification of expressed classical PTPs (PTPome) and the oxidized subset of the PTPome (oxPTPome). We find that mouse and human cells and tissues, including cancer cells, display distinctive PTPomes and oxPTPomes, revealing additional levels of complexity in the regulation of protein-tyrosine phosphorylation in normal and malignant cells.

Src activation involves the coordinated regulation of positive and negative tyrosine phosphorylation sites. The mechanism whereby receptor tyrosine kinases, cytokine receptors, and integrins activate Src is not known. Here, we demonstrate that granulocyte colony-stimulating factor (G-CSF) activates Lyn, the predominant Src kinase in myeloid cells, through Gab2-mediated recruitment of Shp2. After G-CSF stimulation, Lyn dynamically associates with Gab2 in a spatiotemporal manner. The dephosphorylation of phospho-Lyn Tyr507 was abrogated in Shp2-deficient cells transfected with the G-CSF receptor but intact in cells expressing phosphatase-defective Shp2. Auto-phosphorylation of Lyn Tyr396 was impaired in cells treated with Gab2 siRNA. The constitutively activated Shp2E76A directed the dephosphorylation of phospho-Lyn Tyr507 in vitro. Tyr507 did not undergo dephosphorylation in G-CSF-stimulated cells expressing a mutant Gab2 unable to bind Shp2. We propose that Gab2 forms a complex with Lyn and after G-CSF stimulation, Gab2 recruits Shp2, which dephosphorylates phospho-Lyn Tyr507, leading to Lyn activation.