Ayesha N. Shajahan

Human X-box binding protein-1 confers both estrogen independence and antiestrogen resistance in breast cancer cell lines.

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 anti‐apoptotic 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.— Gomez, B. P., Riggins, R. B., Shajahan, A., Klimach, U., Wang, A., Crawford, A. C., Zhu, Y., Zwart, A., Wang, M., Clarke, R. Human X‐Box binding protein‐1 confers both estrogen independence and antiestrogen resis‐tance in breast cancer cell lines. FASEB J. 21, 4013–4027 (2007)

Novel Mechanism of Endothelial Nitric Oxide Synthase Activation Mediated by Caveolae Internalization in Endothelial Cells

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 NG-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-&bgr;-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−/− or caveolin-1−/− 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.

Endoplasmic Reticulum Stress, the Unfolded Protein Response, Autophagy, and the Integrated Regulation of Breast Cancer Cell Fate

How breast cancer cells respond to the stress of endocrine therapies determines whether they will acquire a resistant phenotype or execute a cell-death pathway. After a survival signal is successfully executed, a cell must decide whether it should replicate. How these cell-fate decisions are regulated is unclear, but evidence suggests that the signals that determine 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. The duration of the response to stress is partly mediated by the duration of inositol-requiring enzyme-1 activation following its release from heat shock protein A5. The resulting signals appear to use several B-cell lymphoma-2 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 the regulation of these processes is integrated. Central topologic features of the signaling network that integrate cell-fate regulation and decision execution are discussed.

Glucose-regulated protein 78 controls cross-talk between apoptosis and autophagy to determine antiestrogen responsiveness.

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.

Autophagy and endocrine resistance in breast cancer

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.

Tyrosine phosphorylation-dependence of caveolae-mediated endocytosis.

•u2002 Introduction •u2002 SRC signaling in caveolae‐mediated endocytosis •u2002 Potential role of SRC‐mediated phosphorylation of caveolin‐1 in caveolae‐mediated endocytosis •u2002 Role of actin cytoskeleton in caveolae‐mediated endocytosis •u2002 Conclusion

Gene network signaling in hormone responsiveness modifies apoptosis and autophagy in breast cancer cells.

Resistance to endocrine therapies, whether de novo or acquired, remains a major limitation in the ability to cure many tumors that express detectable levels of the estrogen receptor alpha protein (ER). While several resistance phenotypes have been described, endocrine unresponsiveness in the context of therapy-induced tumor growth appears to be the most prevalent. The signaling that regulates endocrine resistant phenotypes is poorly understood but it involves a complex signaling network with a topology that includes redundant and degenerative features. To be relevant to clinical outcomes, the most pertinent features of this network are those that ultimately affect the endocrine-regulated components of the cell fate and cell proliferation machineries. We show that autophagy, as supported by the endocrine regulation of monodansylcadaverine staining, increased LC3 cleavage, and reduced expression of p62/SQSTM1, plays an important role in breast cancer cells responding to endocrine therapy. We further show that the cell fate machinery includes both apoptotic and autophagic functions that are potentially regulated through integrated signaling that flows through key members of the BCL2 gene family and beclin-1 (BECN1). This signaling links cellular functions in mitochondria and endoplasmic reticulum, the latter as a consequence of induction of the unfolded protein response. We have taken a seed-gene approach to begin extracting critical nodes and edges that represent central signaling events in the endocrine regulation of apoptosis and autophagy. Three seed nodes were identified from global gene or protein expression analyses and supported by subsequent functional studies that established their abilities to affect cell fate. The seed nodes of nuclear factor kappa B (NFkappaB), interferon regulatory factor-1 (IRF1), and X-box binding protein-1 (XBP1)are linked by directional edges that support signal flow through a preliminary network that is grown to include key regulators of their individual function: NEMO/IKKgamma, nucleophosmin and ER respectively. Signaling proceeds through BCL2 gene family members and BECN1 ultimately to regulate cell fate.

Nitric oxide-dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition.

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.

BCL2 and CASP8 regulation by NF-κB differentially affect mitochondrial function and cell fate in antiestrogen-sensitive and -resistant breast cancer cells

Resistance to endocrine therapies remains a major problem in the management of estrogen receptor‐a (ER)‐positive breast cancer. We show that inhibition of NF‐κB (p65/RELA), either by overexpression of a mutant IκB (IκBSR) or a small‐molecule inhibitor of NF‐κB (parthenolide; IC50=500 nM in tamoxifen‐resistant cells), synergistically restores sensitivity to 4‐hydroxytamoxifen (4HT) in resistant MCF7/RR and MCF7/LCC9 cells and further sensitizes MCF‐7 and MCF7/LCC1 control cells to 4HT. These effects are independent of changes in either cell cycle distribution or in the level of autophagy measured by inhibition of p62/SQSTM1 expression and cleavage of LC3. NF‐κB inhibition restores the ability of 4HT to decrease BCL2 expression, increase mitochondrial membrane permeability, and induce a caspase‐dependent apoptotic cell death in resistant cells. Each of these effects is reversed by a caspase 8 (CASP8)‐specific inhibitor that blocks enzyme‐substrate binding. Thus, increased activation of NF‐κB can alter sensitivity to tamoxifen by modulating CASP8 activity, with consequent effects on BCL2 expression, mitochondrial function, and apoptosis. These data provide significant new insights into how molecular signaling affects antiestrogen responsiveness and strongly suggest that a combination of parthenolide and tamoxifen may offer a novel therapeutic approach to the management of some ER‐positive breast cancers.—Nehra, R., Riggins, R. B., Shajahan, A. N., Zwart, A., Crawford, A. C., Clarke, R. BCL2 and CASP8 regulation by NF‐κB differentially affect mitochondrial function and cell fate in antiestrogen‐sensitive and ‐resistant breast cancer cells. FASEB J. 24, 2040–2055 (2010). www.fasebj.org

Caveolin-1 tyrosine phosphorylation enhances paclitaxel-mediated cytotoxicity.

Caveolin-1 (CAV1), a highly conserved membrane-associated protein, is a putative regulator of cellular transformation. CAV1 is localized in the plasmalemma, secretory vesicles, Golgi, mitochondria, and endoplasmic reticulum membrane and associates with the microtubule cytoskeleton. Taxanes such as paclitaxel (Taxol) are potent anti-tumor agents that repress the dynamic instability of microtubules and arrest cells in the G2/M phase. Src phosphorylation of Tyr-14 on CAV1 regulates its cellular localization and function. We report that phosphorylation of CAV1 on Tyr-14 regulates paclitaxel-mediated apoptosis in MCF-7 breast cancer cells. Befitting its role as a multitasking molecule, we show that CAV1 sensitizes cells to apoptosis by regulating cell cycle progression and activation of the apoptotic signaling molecules BCL2, p53, and p21. We demonstrate that phosphorylated CAV1 triggers apoptosis by inactivating BCL2 and increasing mitochondrial permeability more efficiently than non-phosphorylated CAV1. Furthermore, expression of p21, which correlates with taxane sensitivity, is regulated by CAV1 phosphorylation in a p53-dependent manner. Collectively, our findings underscore the importance of CAV1 phosphorylation in apoptosis and suggest that events that negate CAV1 tyrosine phosphorylation may contribute to anti-microtubule drug resistance.