Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae.

Caveolin 1 (Cav1) is a required structural component of caveolae, and its phosphorylation by Src is associated with an increase in caveolae-mediated endocytosis. Here we demonstrate, using quantitative live-cell 4D, TIRF, and FRET imaging, that endocytosis and trafficking of caveolae are associated with a Cav1 Tyr-14 phosphorylation-dependent conformational change, which spatially separates, or loosens, Cav1 molecules within the oligomeric caveolar coat. When tracked by TIRF and spinning-disk microscopy, cells expressing phosphomimicking Cav1 (Y14D) mutant formed vesicles that were greater in number and volume than with Y14F-Cav1-GFP. Furthermore, we observed in HEK cells cotransfected with wild-type, Y14D, or Y14F Cav1-CFP and -YFP constructs that FRET efficiency was greater with Y14F pairs than with Y14D, indicating that pY14-Cav1 regulates the spatial organization of Cav1 molecules within the oligomer. In addition, albumin-induced Src activation or direct activation of Src using a rapamycin-inducible Src construct (RapR-Src) led to an increase in monomeric Cav1 in Western blots, as well as a simultaneous increase in vesicle number and decrease in FRET intensity, indicative of a Src-mediated conformational change in CFP/YFP-tagged WT-Cav1 pairs. We conclude that phosphorylation of Cav1 leads to separation or "spreading" of neighboring negatively charged N-terminal phosphotyrosine residues, promoting swelling of caveolae, followed by their release from the plasma membrane.

BSSV: Bayesian based somatic structural variation identification with whole genome DNA-seq data.

High coverage whole genome DNA-sequencing enables identification of somatic structural variation (SSV) more evident in paired tumor and normal samples. Recent studies show that simultaneous analysis of paired samples provides a better resolution of SSV detection than subtracting shared SVs. However, available tools can neither identify all types of SSVs nor provide any rank information regarding their somatic features. In this paper, we have developed a Bayesian framework, by integrating read alignment information from both tumor and normal samples, called BSSV, to calculate the significance of each SSV. Tested by simulated data, the precision of BSSV is comparable to that of available tools and the false negative rate is significantly lowered. We have also applied this approach to The Cancer Genome Atlas breast cancer data for SSV detection. Many known breast cancer specific mutated genes like RAD51, BRIP1, ER, PGR and PTPRD have been successfully identified.

GX15-070 (obatoclax) induces apoptosis and inhibits cathepsin D- and L-mediated autophagosomal lysis in antiestrogen-resistant breast cancer cells.

In estrogen receptor-positive (ER+) breast cancer cells, BCL2 overexpression contributes to antiestrogen resistance. Direct targeting of the antiapoptotic BCL2 members with GX15-070 (obatoclax), a BH3-mimetic currently in clinical development, is an attractive strategy to overcome antiestrogen resistance in some breast cancers. Recently, GX15-070 has been shown to induce both apoptosis and autophagy, yet the underlying cell death mechanisms have yet to be elucidated. Here, we show that GX15-070 is more effective in reducing the cell density of antiestrogen-resistant breast cancer cells versus sensitive cells and that this increased sensitivity of resistant cells to GX15-070 correlates with an accumulation of autophagic vacuoles. Formation of autophagosomes in GX15-070-treated cells was verified by changes in expression of the lipidation of microtubule-associated protein-1 light chain-3 and both confocal and transmission electron microscopy. While GX15-070 treatment promotes autophagic vacuole and autolysosome formation, p62/SQSTM1, a marker for autophagic degradation, levels accumulate. Moreover, GX15-070 exposure leads to a reduction in cathepsin D (CTSD) and L (CTSL1) protein expression that would otherwise digest autolysosome cargo. Thus, GX15-070 has dual roles in promoting cell death: (i) directly inhibiting antiapoptotic BCL2 family members, thereby inducing apoptosis; and (ii) inhibiting downstream CTSD and CTSL1 protein expression to limit the ability of cells to use degraded material to fuel cellular metabolism and restore homeostasis. Our data highlight a new mechanism of GX15-070-induced cell death that could be used to design novel therapeutic interventions for antiestrogen resistant breast cancer.

Reconstruction of transcriptional regulatory networks by stability-based network component analysis.

Reliable inference of transcription regulatory networks is a challenging task in computational biology. Network component analysis (NCA) has become a powerful scheme to uncover regulatory networks behind complex biological processes. However, the performance of NCA is impaired by the high rate of false connections in binding information. In this paper, we integrate stability analysis with NCA to form a novel scheme, namely stability-based NCA (sNCA), for regulatory network identification. The method mainly addresses the inconsistency between gene expression data and binding motif information. Small perturbations are introduced to prior regulatory network, and the distance among multiple estimated transcript factor (TF) activities is computed to reflect the stability for each TF's binding network. For target gene identification, multivariate regression and t-statistic are used to calculate the significance for each TF-gene connection. Simulation studies are conducted and the experimental results show that sNCA can achieve an improved and robust performance in TF identification as compared to NCA. The approach for target gene identification is also demonstrated to be suitable for identifying true connections between TFs and their target genes. Furthermore, we have successfully applied sNCA to breast cancer data to uncover the role of TFs in regulating endocrine resistance in breast cancer.

Nitrosation-dependent caveolin 1 phosphorylation, ubiquitination, and degradation and its association with idiopathic pulmonary arterial hypertension.

In the present study, we tested the hypothesis that chronic inflammation and oxidative/nitrosative stress induce caveolin 1 (Cav-1) degradation, providing an underlying mechanism of endothelial cell activation/dysfunction and pulmonary vascular remodeling in patients with idiopathic pulmonary arterial hypertension (IPAH). We observed reduced Cav-1 protein despite increased Cav-1 messenger RNA expression and also endothelial nitric oxide synthase (eNOS) hyperphosphorylation in human pulmonary artery endothelial cells (PAECs) from patients with IPAH. In control human lung endothelial cell cultures, tumor necrosis factor α-induced nitric oxide (NO) production and S-nitrosation (SNO) of Cav-1 Cys-156 were associated with Src displacement and activation, Cav-1 Tyr-14 phosphorylation, and destabilization of Cav-1 oligomers within 5 minutes that could be blocked by eNOS or Src inhibition. Prolonged stimulation (72 hours) with NO donor DETANONOate reduced oligomerized and total Cav-1 levels by 40%-80%, similar to that observed in IPAH patient-derived PAECs. NO donor stimulation of endothelial cells for >72 hours, which was associated with sustained Src activation and Cav-1 phosphorylation, ubiquitination, and degradation, was blocked by NOS inhibitor L-NAME, Src inhibitor PP2, and proteosomal inhibitor MG132. Thus, chronic inflammation, sustained eNOS and Src signaling, and Cav-1 degradation may be important causal factors in the development of IPAH by promoting PAEC dysfunction/activation via sustained oxidative/nitrosative stress.

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.

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.

Tyrosine-phosphorylated caveolin-1 (Tyr-14) increases sensitivity to paclitaxel by inhibiting BCL2 and BCLxL proteins via c-Jun N-terminal kinase (JNK).

Paclitaxel, an anti-microtubule agent, is an effective chemotherapeutic drug in breast cancer. Nonetheless, resistance to paclitaxel remains a major clinical challenge. The need to better understand the resistant phenotype and to find biomarkers that could predict tumor response to paclitaxel is evident. In estrogen receptor α-positive (ER(+)) breast cancer cells, phosphorylation of caveolin-1 (CAV1) on Tyr-14 facilitates mitochondrial apoptosis by increasing BCL2 phosphorylation in response to low dose paclitaxel (10 nM). However, two variants of CAV1 exist: the full-length form, CAV1α (wild-type CAV1 or wtCAV1), and a truncated form, CAV1ß. Only wtCAV1 has the Tyr-14 region at the N terminus. The precise cellular functions of CAV1 variants are unknown. We now show that CAV1 variants play distinct roles in paclitaxel-mediated cell death/survival. CAV1ß expression is increased in paclitaxel-resistant cells when compared with sensitive cells. Expression of CAV1ß in sensitive cells significantly reduces their responsiveness to paclitaxel. These activities reflect an essential role for Tyr-14 phosphorylation because wtCAV1 expression, but not a phosphorylation-deficient mutant (Y14F), inactivates BCL2 and BCLxL through activation of c-Jun N-terminal kinase (JNK). MCF-7 cells that express Y14F are resistant to paclitaxel and are resensitized by co-treatment with ABT-737, a BH3-mimetic small molecule inhibitor. Using structural homology modeling, we propose that phosphorylation on Tyr-14 enables a favorable conformation for proteins to bind to the CAV1 scaffolding domain. Thus, we highlight novel roles for CAV1 variants in cell death; wtCAV1 promotes cell death, whereas CAV1ß promotes cell survival by preventing inactivation of BCL2 and BCLxL via JNK in paclitaxel-mediated apoptosis.

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

Endothelial nitric oxide synthase (eNOS)-mediated NO production plays a critical role in the regulation of vascular function and pathophysiology. Caveolin-1 (Cav-1) binding to eNOS holds eNOS in an inactive conformation; however, the mechanism of Cav-1-mediated inhibition of activated eNOS is unclear. Here the role of Src-dependent Cav-1 phosphorylation in eNOS negative feedback regulation is investigated. Using fluorescence resonance energy transfer (FRET) and coimmunoprecipitation analyses, we observed increased interaction between eNOS and Cav-1 following stimulation of endothelial cells with thrombin, vascular endothelial growth factor, and Ca(2+) ionophore A23187, which is corroborated in isolated perfused mouse lung. The eNOS/Cav-1 interaction is blocked by eNOS inhibitor L-N(G)-nitroarginine methyl ester (hydrochloride) and Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3, 4-d] pyrimidine. We also observe increased binding of phosphomimicking Y14D-Cav-1 mutant transduced in human embryonic kidney cells overexpressing eNOS and reduced Ca(2+)-induced NO production compared to cells expressing the phosphodefective Y14F-Cav-1 mutant. Finally, Src FRET biosensor, eNOS small interfering RNA, and NO donor studies demonstrate NO-induced Src activation and Cav-1 phosphorylation at Tyr-14, resulting in increased eNOS/Cav-1 interaction and inhibition of eNOS activity. Taken together, these data suggest that activation of eNOS promotes Src-dependent Cav-1-Tyr-14 phosphorylation and eNOS/Cav-1 binding, that is, eNOS feedback inhibition.

Vitamin E succinate inhibits survivin and induces apoptosis in pancreatic cancer cells.

Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States. Identifying novel chemotherapeutic and chemopreventive approaches is critical in the prevention and treatment of cancers such as pancreatic cancer. Vitamin E succinate (VES) is a redox-silent analog of the fat-soluble vitamin alpha-tocopherol. In the present study, we explored the antiproliferative action of VES and its effects on inhibitor of apoptosis proteins in pancreatic cancer cells. We show that VES inhibits cell proliferation and induces apoptosis in pancreatic cancer cells. Further, we demonstrate that VES downregulates the expression of survivin and X-linked inhibitor of apoptosis proteins. The apoptosis induced by VES was augmented by siRNA-mediated inhibition of survivin in PANC-1 cells. In summary, our results suggest that VES targets survivin signaling and induces apoptosis in pancreatic cancer cells.

Interaction of dietary polyphenols with molecular signaling pathways of antiestrogen resistance: possible role in breast cancer recurrence.

Abstract Breast cancer is the most common cancer diagnosed in women and its global incidence is rising rapidly. Adjuvant hormonal therapy, with antiestrogens (AE) such as tamoxifen and fulvestrant, is highly effective in the treatment of estrogen receptor-positive (ER+) breast cancers and is largely responsible for the increase in survival rates seen in the past four decades. However, nearly 50% of women with ER+ cancer display de novo or acquired resistance to AE therapies. Potential molecular mechanisms driving the resistance phenotype are beginning to be elucidated, allowing further development of more effective therapeutic and preventive strategies to reduce the overall mortality due to breast cancer. Over 70% of breast cancer survivors surveyed report increasing their comsumption of fruits, vegetables, and natural product supplements upon diagnosis. These are rich sources of dietary polyphenols (PPs) that can interact with cell-signaling pathways involved in the development of AE resistance. However, research on mechanisms by which these agents may affect AE resistance and whether PP intake can significantly change breast cancer recurrence is limited. We summarize the available data on the effects of PPs on breast cancer recurrence and the interactions of these compounds with some of the signaling pathways hypothesized to drive cell death and survival involved in the development of AE resistance in breast cancer.

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.

Endoplasmic reticulum stress, the unfolded protein response, and gene network modeling in antiestrogen resistant breast cancer.

Lack of understanding of endocrine resistance remains one of the major challenges for breast cancer researchers, clinicians, and patients. Current reductionist approaches to understanding the molecular signaling driving resistance have offered mostly incremental progress over the past 10 years. As the field of systems biology has begun to mature, the approaches and network modeling tools being developed and applied therein offer a different way to think about how molecular signaling and the regulation of critical cellular functions are integrated. To gain novel insights, we first describe some of the key challenges facing network modeling of endocrine resistance, many of which arise from the properties of the data spaces being studied. We then use activation of the unfolded protein response (UPR) following induction of endoplasmic reticulum stress in breast cancer cells by antiestrogens, to illustrate our approaches to computational modeling. Activation of UPR is a key determinant of cell fate decision making and regulation of autophagy and apoptosis. These initial studies provide insight into a small subnetwork topology obtained using differential dependency network analysis and focused on the UPR gene XBP1. The XBP1 subnetwork topology incorporates BCAR3, BCL2, BIK, NFκB, and other genes as nodes; the connecting edges represent the dependency structures amongst these nodes. As data from ongoing cellular and molecular studies become available, we will build detailed mathematical models of this XBP1-UPR network.

BCL2 and CASP8 regulation by NF-kappaB 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-alpha (ER)-positive breast cancer. We show that inhibition of NF-kappaB (p65/RELA), either by overexpression of a mutant IkappaB (IkappaBSR) or a small-molecule inhibitor of NF-kappaB (parthenolide; IC(50)=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-kappaB 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-kappaB 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.

Co-inhibition of BCL-W and BCL2 restores antiestrogen sensitivity through BECN1 and promotes an autophagy-associated necrosis.

BCL2 family members affect cell fate decisions in breast cancer but the role of BCL-W (BCL2L2) is unknown. We now show the integrated roles of the antiapoptotic BCL-W and BCL2 in affecting responsiveness to the antiestrogen ICI 182,780 (ICI; Fulvestrant Faslodex), using both molecular (siRNA; shRNA) and pharmacologic (YC137) approaches in three breast cancer variants; MCF-7/LCC1 (ICI sensitive), MCF-7/LCC9 (ICI resistant), and LY2 (ICI resistant). YC137 inhibits BCL-W and BCL2 and restores ICI sensitivity in resistant cells. Co-inhibition of BCL-W and BCL2 is both necessary and sufficient to restore sensitivity to ICI, and explains mechanistically the action of YC137. These data implicate functional cooperation and/or redundancy in signaling between BCL-W and BCL2, and suggest that broad BCL2 family member inhibitors will have greater therapeutic value than targeting only individual proteins. Whereas ICI sensitive MCF-7/LCC1 cells undergo increased apoptosis in response to ICI following BCL-W+/-BCL2 co-inhibition, the consequent resensitization of resistant MCF-7/LCC9 and LY2 cells reflects increases in autophagy (LC3 cleavage; p62/SQSTM1 expression) and necrosis but not apoptosis or cell cycle arrest. Thus, de novo sensitive cells and resensitized resistant cells die through different mechanisms. Following BCL-W+BCL2 co-inhibition, suppression of functional autophagy by 3-methyladenine or BECN1 shRNA reduces ICI-induced necrosis but restores the ability of resistant cells to die through apoptosis. These data demonstrate the plasticity of cell fate mechanisms in breast cancer cells in the context of antiestrogen responsiveness. Restoration of ICI sensitivity in resistant cells appears to occur through an increase in autophagy-associated necrosis. BCL-W, BCL2, and BECN1 integrate important functions in determining antiestrogen responsiveness, and the presence of functional autophagy may influence the balance between apoptosis and necrosis.

Changes in mammary caveolin-1 signaling pathways are associated with breast cancer risk in rats exposed to estradiol in utero or during prepuberty.

Developmental stage of rat mammary gland at the time of estrogen exposure determines whether the exposure increases or reduces later breast cancer risk. For example, in utero exposure to 17ß-estradiol (E2) increases, whereas prepubertal exposure to this hormone decreases susceptibility of developing carcinogen-induced mammary tumors. E2 mediates its actions by interacting with caveolin-1 (CAV1), a putative tumor suppressor gene in breast cancer. Mammary tissues from 2-month-old rats exposed to E2 in utero contained decreased levels of CAV1, whereas prepubertal E2 exposure increased the levels, when compared to vehicle controls. Low CAV1 expression was associated with increased cell proliferation and estrogen receptor α expression, and reduced apoptosis in the mammary glands of rats exposed to E2 in utero. In contrast, high CAV1 expression correlated with reduced cell proliferation and cyclin D1 and phospho-Akt levels, and increased apoptosis in the mammary glands of rats exposed to E2 during prepuberty. In support of the role of CAV1 as a negative regulator of a variety of pro-growth signaling proteins, we detected decreased levels of Src and ErbB2 in rats exposed to E2 during prepuberty. Thus, estrogen exposure during mammary gland development affects the expression and function of CAV1 in a manner consistent with observed changes in susceptibility to mammary tumorigenesis.

The role of X-box binding protein-1 in tumorigenicity.

Rapid growth of a tumor can overwhelm the vasculature that supplies it with nutrients and oxygen. Inside such tumors, cells undergo endoplasmic reticulum stress but can survive such adverse microenvironments by an adaptive mechanism called the unfolded protein response (UPR). X-box binding protein-1 (XBP-1) is a critical transcriptional activator of the UPR and is responsible for regulating the function of genes in cell survival. An unconventional splicing of the XBP-1(U) messenger RNA (mRNA) results in two proteins: XBP-1(S) that is often increased in a variety of human cancers and any translated proteins from the unspliced XBP-1(U) mRNA that acts as a dominant negative of endogenous XBP-1(S) action. In cancer cells, overexpression of XBP-1 can confer drug resistance by preventing drug-induced cell-cycle arrest and mitochondrial permeability and apoptosis, while downregulation of XBP-1 increases the sensitivity to killing by hypoxia. XBP-1 is also implicated in cellular de-differentiation, oncovirus infection and the epithelial-to-mesenchymal transition. Given that XBP-1 mediates a wide range of responses in tumorigenesis, it is logical to focus on XBP-1 as an anticancer therapeutic target. Furthermore, combining inhibitors of XBP-1 with other anti-UPR drugs may enhance the activity of some antineoplastic therapies.

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.

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 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.

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 G(2)/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.