While more than 70% of breast cancers express estrogen receptor-α (ER+), endocrine therapies targeting these receptors often fail. The molecular mechanisms that underlie treatment resistance remain unclear. We investigated the potential role of glucose-regulated protein 78 (GRP78) in mediating estrogen resistance. Human breast tumors showed increased GRP78 expression when compared with normal breast tissues. However, GRP78 expression was reduced in ER+ breast tumors compared with HER2-amplifed or triple-negative breast tumors. ER+ antiestrogen-resistant cells and ER+ tumors with an acquired resistant antiestrogen phenotype were both shown to overexpress GRP78, which was not observed in cases of de novo resistance. Knockdown of GRP78 restored antiestrogen sensitivity in resistant cells, and overexpression of GRP78 promoted resistance in sensitive cells. Mechanistically, GRP78 integrated multiple cellular signaling pathways to inhibit apoptosis and stimulate prosurvival autophagy, which was dependent on TSC2/AMPK-mediated mTOR inhibition but not on beclin-1. Inhibition of autophagy prevented GRP78-mediated endocrine resistance, whereas caspase inhibition abrogated the resensitization that resulted from GRP78 loss. Simultaneous knockdown of GRP78 and beclin-1 synergistically restored antiestrogen sensitivity in resistant cells. Together, our findings reveal a novel role for GRP78 in the integration of cellular signaling pathways including the unfolded protein response, apoptosis, and autophagy to determine cell fate in response to antiestrogen therapy.

Albumin transcytosis, a determinant of transendothelial permeability, is mediated by the release of caveolae from the plasma membrane. We addressed the role of Src phosphorylation of the GTPase dynamin-2 in the mechanism of caveolae release and albumin transport. Studies were made in microvascular endothelial cells in which the uptake of cholera toxin subunit B, a marker of caveolae, and 125 I-albumin was used to assess caveolaemediated endocytosis. Albumin binding to the 60-kDa cell surface albumin-binding protein, gp60, induced Src activation (phosphorylation on Tyr 416 ) within 1 min and resulted in Src-dependent tyrosine phosphorylation of dynamin-2, which increased its association with caveolin-1, the caveolae scaffold protein. Expression of kinasedefective Src mutant interfered with the association between dynamin-2, which caveolin-1 and prevented the uptake of albumin. Expression of non-Src-phosphorylatable dynamin (Y231F/Y597F) resulted in reduced association with caveolin-1, and in contrast to WT-dynamin-2, the mutant failed to translocate to the caveolin-rich membrane fraction. The Y231F/Y597F dynamin-2 mutant expression also resulted in impaired albumin and cholera toxin subunit B uptake and reduced transendothelial albumin transport. Thus, Src-mediated phosphorylation of dynamin-2 is an essential requirement for scission of caveolae and the resultant transendothelial transport of albumin.

How breast cancer cells respond to the stress of endocrine therapies determines whether they acquire a resistant phenotype or execute a cell death pathway. A successfully executed survival signal then requires determination of whether or not to replicate. How these cell fate decisions are regulated is unclear but evidence suggests that the signals determining these outcomes are highly integrated. Central to the final cell fate decision is signaling from the unfolded protein response, which can be activated following the sensing of stress within the endoplasmic reticulum. Duration of the response to stress is partly mediated by the duration of inositol requiring enzyme-1 (IRE1; ERN) activation following its release from heat shock protein A5 (HSPA5). The resulting signaling appears to use several B-cell lymphoma-2 (BCL2) family members to both suppress apoptosis and activate autophagy. Changes in metabolism induced by cellular stress are key components of this regulatory system, and further adaptation of the metabolome is affected in response to stress. Here we describe the unfolded protein response, autophagy and apoptosis, and how their regulation is integrated. Central topological features of the signaling network that integrate cell fate regulation and decision execution are discussed.

Human X-box binding protein-1 (XBP1) is an alternatively spliced transcription factor that participates in the unfolded protein response (UPR), a stress-signaling pathway that allows cells to survive the accumulation of unfolded proteins in the endoplasmic reticulum lumen. We have previously demonstrated that XBP1 expression is increased in antiestrogen-resistant breast cancer cell lines and is coexpressed with estrogen receptor alpha (ER) in breast tumors. The purpose of this study is to investigate the role of XBP1 and the UPR in estrogen and antiestrogen responsiveness in breast cancer. Overexpression of spliced XBP1 [XBP1(S)] in ER-positive breast cancer cells leads to estrogen-independent growth and reduced sensitivity to growth inhibition induced by the antiestrogens Tamoxifen and Faslodex in a manner independent of functional p53. Data from gene expression microarray analyses imply that XBP1(S) acts through regulation of the expression of ER, the antiapoptotic gene BCL2, and several other genes associated with control of the cell cycle and apoptosis. Testing this hypothesis, we show that overexpression of XBP1(S) prevents cell cycle arrest and antiestrogen-induced cell death through the mitochondrial apoptotic pathway. XBP1 and/or the UPR may be a useful molecular target for the development of novel predictive and therapeutic strategies in breast cancer.

Abstract-Caveolin-1, the caveolae scaffolding protein, binds to and negatively regulates eNOS activity. As caveolin-1 also regulates caveolae-mediated endocytosis after activation of the 60-kDa albumin-binding glycoprotein gp60 in endothelial cells, we addressed the possibility that endothelial NO synthase (eNOS)-dependent NO production was functionally coupled to caveolae internalization. We observed that gp60-induced activation of endocytosis increased NO production within 2 minutes and up to 20 minutes. NOS inhibitor N G -nitro-L-arginine (L-NNA) prevented the NO production. To determine the role of caveolae internalization in the mechanism of NO production, we expressed dominant-negative dynamin-2 mutant (K44A) or treated cells with methyl-␤-cyclodextrin. Both interventions inhibited caveolae-mediated endocytosis and NO generation induced by gp60. We determined the role of signaling via Src kinase in the observed coupling of endocytosis to eNOS activation. Src activation induced the phosphorylation of caveolin-1, Akt and eNOS, and promoted dissociation of eNOS from caveolin-1. Inhibitors of Src kinase and Akt also prevented NO production. In isolated perfused mouse lungs, gp60 activation induced NO-dependent vasodilation, whereas the response was attenuated in eNOS Ϫ/Ϫ or caveolin-1 Ϫ/Ϫ lungs. Together, these results demonstrate a critical role of caveolae-mediated endocytosis in regulating eNOS activation in endothelial cells and thereby the NO-dependent vasomotor tone. Key Words: caveolin-1 Ⅲ endocytosis Ⅲ vasomotor tone Ⅲ albumin Ⅲ transcytosis E ndothelial NO synthase (eNOS) is modified by N-myristoylation and palmitoylation, which targets the enzyme to caveolae, 1 the plasma membrane cholesterol-rich microdomains. 2,3 Multiple mechanisms are involved in regulating NO production following eNOS activation. eNOS activity is regulated by Ca 2ϩ -calmodulin, phosphorylation activated by kinases such as Src and interactions with caveolin-1, dynamin, and heat shock protein 90 (HSP90). 4 The effects of phosphorylation are complex. Phosphorylation of Ser116 and Thr497 negatively regulates eNOS activity, whereas phosphorylation at Ser635 and Ser1179 has the opposite effect. 5-7 Src kinase activated eNOS by inducing phosphorylation at Tyr83. 8 Phosphorylation at Ser617 functions by affecting the phosphorylation of the above residues. 9 Insulin, estrogen, and shear stress were shown to induce phosphorylationdependent activation of eNOS at Ser1179 independent of increased intracellular [Ca 2ϩ ] 10 -12 eNOS in caveolae is held inactive by its association with caveolin-1, 13 but eNOS activity can be increased by Ca 2ϩ /calmodulin 3 and binding to HSP90 and dynamin-2. 14,15 HSP90 facilitates the phosphorylation of eNOS by forming a ternary complex with eNOS and Akt. 16 Dynamin-2 regulates eNOS activity through the binding of its proline-rich domain to the FAD domain of eNOS, promoting electron transfer between the bound flavins of the reductase domain and increasing NO production. 15 We have shown that activation of t...

The mechanism of caveolin-1–dependent eNOS inactivation is not clear. These studies reveal that NO-mediated Src kinase activation and caveolin-1 phosphorylation promote eNOS binding and inactivation, that is, eNOS negative feedback regulation.

The American Cancer Society estimates that over 200,000 new breast cancer cases are diagnosed annually in the USA alone. Of these cases, the majority are invasive breast cancers and almost 70% are estrogen receptor-α positive. Therapies targeting the estrogen receptor-α are widely applied and include selective estrogen receptor modulators such as tamoxifen, a selective estrogen receptor downregulator such as Fulvestrant (Faslodex; FAS, ICI 182,780), or one of the third-generation aromatase inhibitors including letrozole or anastrozole. While these treatments reduce breast cancer mortality, many estrogen receptor-α-positive tumors eventually recur, highlighting the clinical significance of endocrine therapy resistance. The signaling leading to endocrine therapy resistance is poorly understood; however, preclinical studies have established an important role for autophagy in the acquired resistance phenotype. Autophagy is a cellular degradation process initiated in response to stress or nutrient deprivation, which attempts to restore metabolic homeostasis through the catabolic lysis of aggregated proteins, unfolded/misfolded proteins or damaged subcellular organelles. The duality of autophagy, which can be either pro-survival or pro-death, is well known. However, in the context of endocrine therapy resistance in breast cancer, the inhibition of autophagy can potentiate resensitization of previously antiestrogen resistant breast cancer cells. In this article, we discuss the complex and occasionally contradictory roles of autophagy in cancer and in resistance to endocrine therapies in breast cancer.

Caveolae-mediated endocytosis in endothelial cells is stimulated by the binding of albumin to gp60, a specific albumin-binding protein localized in caveolae. The activation of gp60 induces its cell surface clustering and association with caveolin-1, the caveolar-scaffolding protein. This interaction leads to G i -induced Src kinase activation, which in turn signals dynamin-2-mediated fission and directed migration of caveolaederived vesicles from apical to basal membrane. In this study, we investigated the possible role of the G␤␥ heterodimer in signaling G i -induced Src activation and subsequent caveolae-mediated endocytosis. We observed using rat lung microvascular endothelial cells that expression of the C terminus of ␤-adrenergic receptor kinase (ct-␤ARK), an inhibitor G␤␥ signaling, prevented gp60-dependent Src activation as well as caveolae-mediated endocytosis and transcellular transport of albumin and uptake of cholera toxin subunit B, a specific marker of caveolae internalization. Expression of ct-␤ARK also prevented Src-mediated tyrosine phosphorylation of caveolin-1 and dynamin-2 and the resultant phosphorylation-dependent association of dynamin-2 and caveolin-1. Also, the direct activation of G␤␥ using a specific cell-permeant activating peptide (myristoylated-SIRKALNILGYPDYD) simulated the effects of gp60 in inducing Src activation, caveolin-1, and dynamin-2 phosphorylation as well as caveolae-mediated endocytosis of cholera toxin subunit B. The myristoylated-SIRKALNILGYPDYD peptide-induced responses were inhibited by the expression of ct-␤ARK. Taken together, our results demonstrate that G␤␥ activation of Src signals caveolaemediated endocytosis and transendothelial albumin transport via transcytosis.