Focal adhesion kinase (FAK) has been implicated in the development of cancers, including those of the breast. Nevertheless, the molecular and cellular mechanisms by which FAK promotes mammary tumorigenesis in vivo are not well understood. Here, we show that targeted deletion of FAK in mouse mammary epithelium significantly suppresses mammary tumorigenesis in a well-characterized breast cancer model. Ablation of FAK leads to the depletion of a subset of bipotent cells in the tumor that express both luminal marker keratin 8/18 and basal marker keratin 5. Using mammary stem/progenitor markers, including aldehyde dehydrogenase, CD24, CD29, and CD61, we further revealed that ablation of FAK reduced the pool of cancer stem/progenitor cells in primary tumors of FAK-targeted mice and impaired their selfrenewal and migration in vitro. Finally, through transplantation in NOD-SCID mice, we found that cancer stem/progenitor cells isolated from FAK-targeted mice have compromised tumorigenicity and impaired maintenance in vivo. Together, these results show a novel function of FAK in maintaining the mammary cancer stem/progenitor cell population and provide a novel mechanism by which FAK may promote breast cancer development and progression. [Cancer Res 2009;69(2):466-74]

Opiate abuse causes adaptive changes in several processes of synaptic transmission in which the glutamatergic system appears a critical element involved in opiate tolerance and dependence, but the underlying mechanisms remain unclear. In the present study, we found that glutamate uptake in hippocampal synaptosomes was significantly increased (by 70% in chronic morphine-treated rats) during the morphine withdrawal period, likely attributable to an increase in the number of functional glutamate transporters. Immunoblot analysis showed that expression of GLT1 (glutamate transporter subtype 1) was identified to be upregulated in synaptosomes but not in total tissues, suggesting a redistribution of glutamate transporter expression. Moreover, the increase in glutamate uptake was reproduced in cultured neurons during morphine withdrawal, and the increase of uptake in neurons could be blocked by dihydrokainate, a specific inhibitor of GLT1. Cell surface biotinylation and immunoblot analysis showed that morphine withdrawal produced an increase in GLT1 expression rather than EAAC1 (excitatory amino acids carrier 1), a neuronal subtype, at the cultured neuronal cell surface, whereas no significant change was observed in that of cultured astrocytes. Electron microscopy also revealed that GLT1 expression was markedly increased in the nerve terminals of hippocampus and associated with the plasma membrane in vivo. These results suggest that GLT1 in hippocampal neurons can be induced to translocate to the nerve terminals and express on the cell surface during morphine withdrawal. The translocation of GLT1 at synapses during morphine withdrawal provides a neuronal mechanism for modulation of excitatory neurotransmission during opiate abuse.

Rab1a is a member of the Rab family of small GTPases with a well characterized function in the regulation of vesicle trafficking from the endoplasmic reticulum to the Golgi apparatus and within Golgi compartments. The integrin family heterodimeric transmembrane proteins serve as major receptors for extracellular matrix proteins, which play essential roles in cell adhesion and migration. Although effects on intracellular trafficking of integrins or other key cargos by Rab1a could influence cell migration, the regulatory mechanisms linking Rab1a to cell migration are not well understood. Here, we report identification of Rab1a as a novel regulator of cell migration using an unbiased RNAi screen targeting GTPases. Inhibition of Rab1a reduced integrin-mediated cell adhesion and spreading on fibronectins, reduced integrin ␤1 localization to lipid rafts, and decreased recycling of integrin ␤1 to the plasma membrane. Analysis of Rab1a effector molecules showed that p115 mediated Rab1a regulation of integrin recycling and lipid raft localization in cell migration. Taken together, these results suggest a novel function for Rab1a in the regulation of cell migration through controlling integrin ␤1 recycling and localization to lipid rafts via a specific downstream effector pathway.

Background: FAK has both kinase and scaffolding functions. Results: Disruption of the function of FAK scaffolding to mediate endophilin A2 phosphorylation inhibits mammary tumor growth and metastasis in vivo by decreasing tumor cell markers for EMT and their MaCSCs activities. Conclusion:The function of FAK scaffolding is important for promoting mammary tumor progression. Significance: Targeting the scaffolding function of FAK may be important in breast cancer therapy.

Cancer stem cells (CSCs) 2 are proposed to play important roles in the initiation and progression of breast and several other cancers recently (1-4). According to the CSC concept, although the conventional therapies could destroy the bulk of the tumor mass, even a small amount of residual CSCs could lead to recurrence of the cancer due to their stem cell-like ability for self-renewal and differentiation (2). Several recent studies also suggested that CSCs, such as mammary cancer stem cells (MaCSCs), are more resistant to conventional cancer therapies compared with the bulk of cells in the tumor mass (5-9), which could further decrease the effectiveness of conventional cancer treatment strategies. Therefore, the characterization of key signaling molecules and pathways that regulate MaCSCs will be important for understanding mammary carcinogenesis and development of more effective therapeutic strategies for breast cancer through targeting MaCSCs.Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that plays a major role in mediating signal transduction by integrins as well as growth factor receptors in the regulation of cell adhesion, migration, survival, proliferation, and differentiation in a variety of cells (10 -14). Upon its activation by integrin-mediated cell adhesion or other stimuli, FAK undergoes autophosphorylation at Tyr 397 to create a binding site for several Src homology 2 domain-containing molecules, including Src (15, 16) and the p85 subunit of PI3K (17, 18). FAK association and activation of PI3K through autophosphorylated Tyr 397 leads to increased production of 3Ј-phosphorylated phospholipid (17), which activates Akt to promote cell survival by regulating several other proteins (19 -23). Consistent with its role in regulation of multiple signaling pathways, FAK has been implicated in the development of breast cancer and other malignancies (24,25). Recent studies by several groups, including us, showed that ablation of FAK suppressed mammary tumorigenesis and progression in mouse models of breast cancer, demonstrating directly a causal role of FAK in promoting breast cancer in vivo (26 -29). Moreover, using a combination of well characterized markers and tumorsphere formation assays (30 -34) as well as transplantation experiments, our previous study revealed that inactivation of FAK reduced MaCSCs in primary tumors developed in FAK conditional KO mice, decreased their self-renewal and migration in vitro, and compromised their tumorigenicity and maintenance in vivo (28). These results suggest that deletion of FAK may suppress mammary tumorigenesis and progression by affecting MaCSCs. Nevertheless, it is not clear which of the multiple FAK downstream signaling pathways mediate its regulation of MaCSCs. The potential mechanisms that allow mammary tumorigenesis and metastasis in FAK conditional KO mice, albeit at a reduced level, are not well understood.Pyk2 is the other member of the FAK subfamily cytoplasmic tyrosine kinases that shares significant sequence homology and

Neurotransmitter ␥-aminobutyric acid (GABA) release to the synaptic clefts is mediated by the formation of a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, which includes two target SNAREs syntaxin 1A and SNAP-25 and one vesicle SNARE VAMP-2. The target SNAREs syntaxin 1A and SNAP-25 form a heterodimer, the putative intermediate of the SNARE complex. Neurotransmitter GABA clearance from synaptic clefts is carried out by the reuptake function of its transporters to terminate the postsynaptic signaling. Syntaxin 1A directly binds to the neuronal GABA transporter GAT-1 and inhibits its reuptake function. However, whether other SNARE proteins or SNARE complex regulates GABA reuptake remains unknown. Here we demonstrate that SNAP-25 efficiently inhibits GAT-1 reuptake function in the presence of syntaxin 1A. This inhibition depends on SNAP-25/syntaxin 1A complex formation. The H3 domain of syntaxin 1A is identified as the binding sites for both SNAP-25 and GAT-1. SNAP-25 binding to syntaxin 1A greatly potentiates the physical interaction of syntaxin 1A with GAT-1 and significantly enhances the syntaxin 1A-mediated inhibition of GAT-1 reuptake function. Furthermore, nitric oxide, which promotes SNAP-25 binding to syntaxin 1A to form the SNARE complex, also potentiates the interaction of syntaxin 1A with GAT-1 and suppresses GABA reuptake by GAT-1. Thus our findings delineate a further molecular mechanism for the regulation of GABA reuptake by a target SNARE complex and suggest a direct coordination between GABA release and reuptake. ␥-Aminobutyric acid (GABA)3 is the major inhibitory neurotransmitter in the central nervous system, which is released from the presynaptic terminals through the docking and fusion of synaptic vesicles with the plasma membrane. Membrane fusion and subsequent GABA release are catalyzed by the assembly of a ternary complex from soluble N-ethylmaleimidesensitive factor attachment protein receptor (SNARE) proteins (1). The ternary complex is composed of two plasma membrane proteins, including syntaxin 1A and synaptosomal associated protein of 25 kDa (SNAP-25), which are called target SNAREs (t-SNAREs), and one vesicle-associated protein synaptobrevin 2 (VAMP-2), which is called vesicle SNARE (2, 3). The t-SNAREs syntaxin 1A and SNAP-25 form a heterodimer, the putative intermediate of the SNARE complex, and offer target sites for the vesicle SNARE VAMP-2 leading to membrane fusion (4, 5). After membrane fusion, GABA is released from synaptic vesicles to synaptic clefts and binds to postsynaptic GABA receptors and thus transmits the signal to the postsynaptic terminals.GABA is cleared away rapidly from synaptic clefts to terminate synaptic transmission through the reuptake function of its specific, high affinity, sodium-and chloride-dependent transporters (6), which are located on presynaptic terminals and surrounding glial cells (7). GABA transporters are mainly divided into four subtypes, including GAT-1, GAT-2, GAT-3, and BGT-1, of which GAT-1 is the mos...

Objective To investigate the prescription rate of short-term systemic use of glucocorticoids during hospitalization in patients with cardiogenic shock (CS), and outcomes related with glucocorticoid use. Methods We extracted patients' information from the Medical Information Mart for Intensive Care IV version 2.0 (MIMIC-IV v2.0) database. The primary endpoint was 90-day all-cause mortality. Secondary safety endpoints were infection identified by bacterial culture and at least one episode of hyperglycemia after ICU admission. Propensity score matching (PSM) was used to balance baseline characteristics. The difference in cumulative mortality rate between these treated with and without glucocorticoids was assessed by Kaplan–Meier curve with log-rank test. Independent risk factors for endpoints were identified by Cox or Logistic regression analysis. Results A total of 1528 patients were enrolled, and one-sixth of these patients received short-term systemic therapy of glucocorticoids during hospitalization. These conditions, including rapid heart rate, the presence of rheumatic disease, chronic pulmonary disease and septic shock, high lactate level, the requirements of mechanical ventilation and continuous renal replacement therapy, were associated with an increase in glucocorticoid administration (all P ≤ 0.024). During a follow-up of 90 days, the cumulative mortality rate in patients treated with glucocorticoids was significantly higher than that in these untreated with glucocorticoids (log-rank test, P < 0.001). Multivariable Cox regression analysis showed that glucocorticoid use (hazard ratio 1.48, 95% confidence interval [CI] 1.22–1.81; P < 0.001) was independently associated with an increased risk for 90-day all-cause mortality. This result was consistent irrespective of age, gender, the presence of myocardial infarction, acute decompensated heart failure and septic shock, and inotrope therapy, but was more evident in low-risk patients as assessed by ICU scoring systems. Additionally, multivariable Logistic regression analysis showed that glucocorticoid exposure was an independent predictor of hyperglycemia (odds ratio 2.14, 95% CI 1.48–3.10; P < 0.001), but not infection (odds ratio 1.23, 95% CI 0.88–1.73; P = 0.221). After PSM, glucocorticoid therapy was also significantly related with increased risks of 90-day mortality and hyperglycemia. Conclusions Real-world data showed that short-term systemic use of glucocorticoids was common in CS patients. Importantly, these prescriptions were associated with increased risks of adverse events.

Supplementary Table 1 from Mammary Epithelial-Specific Ablation of the Focal Adhesion Kinase Suppresses Mammary Tumorigenesis by Affecting Mammary Cancer Stem/Progenitor Cells