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Effective knockdown of PPARγ at RNA levels in LTED breast cancer cell lines. A, MCF-7:5C cells were transfected with scrambled siRNA or specific PPARγ siRNA for 72 hours. Next, cells were treated with a vehicle control (0.1% EtOH) or E2 (1nM) for 48 hours. Cells were then harvested in TRIzol reagent. PPARγ expression levels were quantitated using RT-PCR. **P

Peroxisome proliferator-activated receptor γ (PPARγ) is an important transcription factor that modulates lipid metabolism and inflammation. However, it remains unclear whether PPARγ is involved in modulation of estrogen (E2)-induced inflammation, thus affecting apoptosis of E2-deprived breast cancer cells, MCF-7:5C and MCF-7:2A. Here, we demonstrated that E2 treatment suppressed the function of PPARγ in both cell lines, although the suppressive effect in MCF-7:2A cells was delayed owing to high PPARγ expression. Activation of PPARγ by a specific agonist, pioglitazone, selectively blocked the induction of TNFα expression by E2, but did not affect other adipose inflammatory genes, such as fatty acid desaturase 1 and IL6. This suppression of TNFα expression by pioglitazone was mainly mediated by transrepression of nuclear factor-κB (NF-κB) DNA-binding activity. A novel finding was that NF-κB functions as an oxidative stress inducer in MCF-7:5C cells but an antioxidant in MCF-7:2A cells. Therefore, the NF-κB inhibitor JSH-23 displayed effects equivalent to those of pioglitazone, with complete inhibition of apoptosis in MCF-7:5C cells, but it increased E2-induced apoptosis in MCF-7:2A cells. Depletion of PPARγ by siRNA or the PPARγ antagonist T0070907 accelerated E2-induced apoptosis, with activation of NF-κB–dependent TNFα and oxidative stress. For the first time, we demonstrated that PPARγ is a growth signal and has potential to modulate NF-κB activity and oxidative stress in E2-deprived breast cancer cell lines. All of these findings suggest that anti-PPARγ therapy is a novel strategy to improve the therapeutic effects of E2-induced apoptosis in E2-deprived breast cancer.

Supplementary Figure 1 from The G Protein–Coupled Receptor GPR30 Inhibits Proliferation of Estrogen Receptor–Positive Breast Cancer Cells

Supplementary Methods from The G Protein–Coupled Receptor GPR30 Inhibits Proliferation of Estrogen Receptor–Positive Breast Cancer Cells

How cancer cells adapt to evade the therapeutic effects of drugs targeting oncogenic drivers is poorly understood. Here we report an epigenetic mechanism leading to the adaptive resistance of triple-negative breast cancer (TNBC) to fibroblast growth factor receptor (FGFR) inhibitors. Prolonged FGFR inhibition suppresses the function of BRG1-dependent chromatin remodelling, leading to an epigenetic state that derepresses YAP-associated enhancers. These chromatin changes induce the expression of several amino acid transporters, resulting in increased intracellular levels of specific amino acids that reactivate mTORC1. Consistent with this mechanism, addition of mTORC1 or YAP inhibitors to FGFR blockade synergistically attenuated the growth of TNBC patient-derived xenograft models. Collectively, these findings reveal a feedback loop involving an epigenetic state transition and metabolic reprogramming that leads to adaptive therapeutic resistance and provides potential therapeutic strategies to overcome this mechanism of resistance. Li et al. define an adaptive resistance mechanism against FGFR inhibitor treatment in breast cancer attributed to loss of BRG1 chromatin recruitment, reactivation of YAP-dependent enhancers and upregulation of amino acid-induced mTORC1 activity.

Our clinically relevant finding is that glucocorticoids block estrogen (E(2))-induced apoptosis in long-term E(2)-deprived (LTED) breast cancer cells. However, the mechanism remains unclear. Here, we demonstrated that E(2) widely activated adipose inflammatory factors such as fatty acid desaturase 1 (FADS1), interleukin-6 (IL-6), and tumor necrosis factor α (TNFα) in LTED breast cancer cells. Activation of glucocorticoid receptor (GR) by the synthetic glucocorticoid dexamethasone (Dex) upregulated FADS1 and IL-6 but downregulated TNFα expression. Furthermore, Dex was synergistic or additive with E(2) in upregulating FADS1 and IL-6 expression, whereas it selectively and constantly suppressed TNFα expression induced by E(2) in LTED breast cancer cells. Regarding regulation of endoplasmic reticulum stress, Dex effectively blocked activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) by E(2), but it had no inhibitory effects on inositol-requiring protein 1 alpha (IRE1α) expression increased by E(2). Consistently, results from reverse-phase protein array (RPPA) analysis demonstrated that Dex could not reverse IRE1α-mediated degradation of phosphoinositide 3-kinase (PI3K)/Akt-associated signal pathways activated by E(2). Unexpectedly, activated GR preferentially repressed nuclear factor-κB (NF-κB) DNA-binding activity and expression of NF-κB-dependent gene TNFα induced by E(2), leading to the blockade of E(2)-induced apoptosis. Together, these data suggest that trans-suppression of NF-κB by GR in the nucleus is a fundamental mechanism thereby blocking E(2)-induced apoptosis in LTED breast cancer cells. This study provided an important rationale for restricting the clinical use of glucocorticoids, which will undermine the beneficial effects of E(2)-induced apoptosis in aromatase inhibitor-resistant breast cancer patients.

How cancer cells adapt to evade the therapeutic effects of drugs targeting oncogenic drivers is poorly understood. Here we report an epigenetic mechanism leading to the adaptive resistance of triple-negative breast cancer (TNBC) to fibroblast growth factor receptor (FGFR) inhibitors. Prolonged FGFR inhibition suppresses the function of BRG1-dependent chromatin remodeling leading to an epigenetic state that derepresses YAP-associated enhancers. These chromatin changes induce the expression of several amino acid transporters resulting in increased intracellular levels of specific amino acids that reactivate mTORC1. Collectively, these findings reveal a novel feedback loop involving an epigenetic state transition and metabolic reprogramming that leads to adaptive therapeutic resistance.

Brain metastases (BM) are a leading cause of cancer death and prognosis remains poor despite treatment advances at other sites. Models are central to therapeutic development, but few orthotopic patient-derived xenograft (PDX) models of BM exist. To represent diversity across BM types, we established a program to create orthotopic PDX at scale from all BM patients. To date BM were received from 100 patients and PDX attempted by direct brain injection (PDX, n=89) or injection of low passage patient-derived cell lines (PDCLX, n=11). We created 65 successful BM PDX from 13 cancers: 17 lung (55% take), 15 breast (68%), 6 melanoma (75%), 5 CNS lymphoma (83%), 3 gastrointestinal (75%), 2 esophageal (40%), 2 ovarian (67%), 1 sarcoma (100%), 1 laryngeal (100%), 1 prostate (100%), 1 pancreatic (100%), 1 uterine adenosarcoma (100%), and 1 yolk sac tumor (100%). Take rate was similar for models derived from patients with prior chemotherapy-only versus immune/targeted therapy-only (63 vs 58%). Fifteen patients had live tumor and matching PBMCs archived for modeling in vitro immunotherapy responses. Mean time to moribund among different cancer types ranged from 27 days (yolk sac tumor) to 177.5 days (ovarian). BM PDX had a favorable timeline for preclinical study (90% moribund at 180 days). All PDX retained high fidelity to the patient driver SNVs and copy aberrations, even at >P4. No significant differences noted by immunodeficient strain (SCID versus NSG) or injection site (orthotopic versus heterotopic). Explants from BM PDX were able to generate long-term cell lines (60%) or short-term cultures with qualitative concordance of model-to-patient responses to targeted therapy (Osimertinib, EGFRi) and immunotherapy (Pembrolizumab, PD1i). Genomic and clinical data were used to create the DFCI BM PDX cBioPortal for public release and models distribution will be available through the DFCI Center for Patient Derived Models.

Lymphomas of the central nervous system (CNSL) are rare tumors with few treatment options; however, recent genomic studies have uncovered several druggable targets. To facilitate novel treatment discovery, we sought to establish patient-derived xenograft (PDX) models and cell lines from patients with newly diagnosed or recurrent CNSL. PDX models were attempted from fresh tumor samples from 18 consented patients for whom rich clinical annotation was available. Orthotopic intracranial injections were performed via stereotactic injection. Cell line generation was attempted from both primary tumors (PDCL) and PDX samples (PDXCL). Characterization included histopathology, targeted exome sequencing, low-pass whole-genome sequencing for copy number evaluation, and expression profiling of matched patient tumors and PDXs. Drug response testing was performed using CellTiterGlo. From 18 samples attempted, 8 PDX models were successfully generated: 2 primary CNS diffuse large B-cell lymphoma (DLBCL), 5 secondary CNS DLBCL, and 1 secondary cutaneous T-cell lymphoma (CTCL). Mean time to moribund was 66 days. All 8 PDXs grew successfully as orthotopic models, and 4 also grew in subrenal capsule. Additionally, 6 PDCL and 2 PDXCL were established (> P3). Models faithfully represented primaries, showing nearly identical histopathology, immunoprofiles, and genomic signatures. To support expanded and rapid preclinical use of these models in drug testing we documented that PDX could be explanted to create short-term cell preparations or long-term cell lines with a 100% and 40% success rate, respectively. Proof of principle testing showed that explanted DLBCL PDX cells and cell lines showed sensitivity to copanlisib and venetoclax, which were not efficacious in the CTCL PDX. These findings showcase a diverse collection of CNSL models demonstrating high fidelity to primary tumors at the genomic and phenotypic levels, emphasizing their utility for preclinical studies and as patient avatars to rapidly determine sensitivities to existing and novel regimens prior to initiation of therapy.

Brain metastases (BM) are a leading cause of cancer death and prognosis remains poor despite treatment advances at other sites. Models are central to therapeutic development, but few orthotopic patient-derived xenograft (PDX) models of BM exist. To represent diversity across BM types, we established a program to create orthotopic PDX at scale from all BM patients. To date BM were received from 100 patients and PDX attempted by direct brain injection (PDX, n=89) or injection of low passage patient-derived cell lines (PDCLX, n=11). We created 65 successful BM PDX from 13 cancers: 17 lung (55% take), 15 breast (68%), 6 melanoma (75%), 5 CNS lymphoma (83%), 3 gastrointestinal (75%), 2 esophageal (40%), 2 ovarian (67%), 1 sarcoma (100%), 1 laryngeal (100%), 1 prostate (100%), 1 pancreatic (100%), 1 uterine adenosarcoma (100%), and 1 yolk sac tumor (100%). Take rate was similar for models derived from patients with prior chemotherapy-only versus immune/targeted therapy-only (63 vs 58%). Fifteen patients had live tumor and matching PBMCs archived for modeling in vitro immunotherapy responses. Mean time to moribund among different cancer types ranged from 27 days (yolk sac tumor) to 177.5 days (ovarian). BM PDX had a favorable timeline for preclinical study (90% moribund at 180 days). All PDX matched the patient driver SNVs and copy aberrations, even at >P4. No significant differences noted by immunodeficient strain (SCID versus NSG) or injection site (orthotopic versus heterotopic). Explants from BM PDX were able to generate long-term cell lines (60%) or short-term cultures with qualitative concordance of model-to-patient responses to targeted therapy (Osimertinib, EGFRi) and immunotherapy (Pembrolizumab, PD1i). Genomic and clinical data were used to create the DFCI BM PDX cBioPortal for public release and models distribution will be available through the DFCI Center for Patient Derived Models.

Peroxisome proliferator-activated receptor γ (PPARγ) is an important transcription factor that modulates lipid metabolism and inflammation. However, it remains unclear whether PPARγ is involved in modulation of estrogen (E(2))-induced inflammation, thus affecting apoptosis of E(2)-deprived breast cancer cells, MCF-7:5C and MCF-7:2A. Here, we demonstrated that E(2) treatment suppressed the function of PPARγ in both cell lines, although the suppressive effect in MCF-7:2A cells was delayed owing to high PPARγ expression. Activation of PPARγ by a specific agonist, pioglitazone selectively blocked the induction of tumor necrosis factor alpha (TNFα) expression by E(2), but did not affect other adipose inflammatory genes, such as fatty acid desaturase 1(FADS1) and interleukin-6 (IL-6). This suppression of TNFα expression by pioglitazone was mainly mediated by trans-repression of nuclear factor-κB (NF-κB) DNA-binding activity. A novel finding was that NF-κB functions as an oxidative stress inducer in MCF-7:5C cells but an antioxidant in MCF-7:2A cells. Therefore, the NF-κB inhibitor JSH-23 displayed effects equivalent to those of pioglitazone, with complete inhibition of apoptosis in MCF-7:5C cells, but it increased E(2)-induced apoptosis in MCF-7:2A cells. Depletion of PPARγ by small interfering RNA or the PPARγ antagonist T0070907 accelerated E(2)-induced apoptosis, with activation of NF-κB-dependent TNFα and oxidative stress. For the first time, we demonstrated that PPARγ is a growth signal and has potential to modulate NF-κB activity and oxidative stress in E(2)-deprived breast cancer cell lines. All of these findings suggest that anti-PPARγ therapy is a novel strategy to improve the therapeutic effects of E(2)-induced apoptosis in E(2)-deprived breast cancer.

The nuclear pore complex subunit TPR is found in at least five different oncogenic fusion kinases, including TPR-MET, yet how TPR fusions promote activation of kinases and their oncogenic activities remains poorly understood. Here we report the crystal structure of TPR(2-142), the MET fusion partner of oncogenic TPR-MET. TPR(2-142) contains a continuous 124-residue α helix that forms an antiparallel tetramer from two leucine zipper-containing parallel coiled coils. Remarkably, single mutations cause strikingly different conformations of the coiled coil, indicating its highly dynamic nature. We further show that fusion of TPR(2-142) to the MET intracellular domain strongly and selectively stabilizes the αG helix of the MET kinase domain, and mutations of only the TPR leucine zipper residues at the junction to MET, but not other leucine zipper residues, abolish kinase activation. Together, these results provide critical insight into the TPR structure and its ability to induce dimerization and activation of fusion kinases.

In laboratory studies, acquired resistance to long-term antihormonal therapy in breast cancer evolves through two phases over 5 y. Phase I develops within 1 y, and tumor growth occurs with either 17β-estradiol (E 2 ) or tamoxifen. Phase II resistance develops after 5 y of therapy, and tamoxifen still stimulates growth; however, E 2 paradoxically induces apoptosis. This finding is the basis for the clinical use of estrogen to treat advanced antihormone-resistant breast cancer. We interrogated E 2 -induced apoptosis by analysis of gene expression across time (2–96 h) in MCF-7 cell variants that were estrogen-dependent (WS8) or resistant to estrogen deprivation and refractory (2A) or sensitive (5C) to E 2 -induced apoptosis. We developed a method termed differential area under the curve analysis that identified genes uniquely regulated by E 2 in 5C cells compared with both WS8 and 2A cells and hence, were associated with E 2 -induced apoptosis. Estrogen signaling, endoplasmic reticulum stress (ERS), and inflammatory response genes were overrepresented among the 5C-specific genes. The identified ERS genes indicated that E 2 inhibited protein folding, translation, and fatty acid synthesis. Meanwhile, the ERS-associated apoptotic genes Bcl-2 interacting mediator of cell death (BIM; BCL2L11) and caspase-4 (CASP4), among others, were induced. Evaluation of a caspase peptide inhibitor panel showed that the CASP4 inhibitor z-LEVD-fmk was the most active at blocking E 2 -induced apoptosis. Furthermore, z-LEVD-fmk completely prevented poly (ADP-ribose) polymerase (PARP) cleavage, E 2 -inhibited growth, and apoptotic morphology. The up-regulated proinflammatory genes included IL, IFN, and arachidonic acid-related genes. Functional testing showed that arachidonic acid and E 2 interacted to superadditively induce apoptosis. Therefore, these data indicate that E 2 induced apoptosis through ERS and inflammatory responses in advanced antihormone-resistant breast cancer.

The SNF2-related CBP activator protein (SRCAP) serves as a coactivator for several nuclear receptors including the androgen receptor (AR). SRCAP is an ATPase that is the core subunit of a large multiprotein complex and was shown to incorporate the histone variant H2A.Z into nucleosomes. In this report, we demonstrate that SRCAP is expressed in the epithelium of normal prostate and in prostate carcinoma cells, and is associated with AR in the nucleus. Using transient transfection assays we demonstrate that SRCAP activates hormone-dependent transcription of the androgen responsive, prostate specific antigen (PSA)-Luciferase reporter gene in human prostate cells. The in vivo occupancy of SRCAP at the endogenous PSA promoter is demonstrated using chromatin immunoprecipitation assays. ShRNA mediated knockdown of SRCAP resulted in decreased H2A.Z binding at the enhancer region of the PSA promoter and decreased expression of PSA in prostate cancer cells. Furthermore, inhibition of SRCAP expression significantly inhibited androgen dependent prostate cancer cell growth. These data identify SRCAP as a physiologically relevant mediator of PSA expression, and demonstrate that SRCAP plays a role in prostate cancer cell proliferation.

Major operative injury initiates immunologic changes that may result in a systemic inflammatory response, leading to organ dysfunction/sepsis. Catecholamines seem to be key mediators. The effects of beta(2) receptor blockade in vitro and in vivo before and after operative injury were studied to clarify their role.In vitro studies were done in RAW 264.7 cells using epinephrine (50 micromol/L) with or without alpha(2)- and beta(2)-receptor blockade. Comparative gene expression analysis on the Toll-like receptor (TLR)-4 receptor signaling pathway was performed between RAW cells pretreated with epinephrine with subsequent lipopolysaccharide (LPS) stimulation versus LPS alone. Confirmatory studies were performed by real-time reverse transcription polymerase chain reaction (RT-PCR). Tumor necrosis factor (TNF)-alpha gene and protein expression were determined via real time RT-PCR and enzyme-linked immunosorbent assay, respectively. In vivo, Balb/C mice received no treatment nor injury (group I). Group II received operative injury and vehicle injection, group III received ICI 118,551 (beta(2)-receptor antagonist) 30 minutes before or in separate studies after operative injury. At 7 days, splenic macrophages were harvested and cytokine production was measured with or without LPS. In separate experiments, cecal ligation and puncture (CLP) was done 7 days after operation and survival determined.In vitro studies demonstrated that epinephrine pretreatment significantly increased TNF-alpha production with LPS stimulation (P.05). beta(2)-Receptor blockade significantly attenuated (P.05) LPS stimulated TNF-alpha production. MD-2, an essential coactivator of TLR-4 signaling, gene expression was significantly elevated when cells were pretreated with epinephrine before LPS exposure (P.001). In vivo studies demonstrated a significant decrease (P.05) in TNF-alpha and interleukin-6 production in the ICI 118,551 group. Similar findings were demonstrated measuring monocyte chemoattractant protein-1 and interferon-gamma cytokine levels (P.05) versus no treatment. ICI 118,551 treatment 30 minutes before operation demonstrated a 31% reduction in mortality after CLP (P.05).This study demonstrates that beta(2)-receptor blockade reduces macrophage cytokine production and improves survival showing the critical importance of catecholamines to the immunologic response in surgery.

Background Ovarian cancer is the most common cause of cancer related death from gynecologic tumors in the United States. The insidious nature of the disease precludes early diagnosis, therefore surgical debulking and chemotherapy are considered as standard treatment modalities for advanced stages. We investigated the effect of the LXR agonist, T0901317, on ovarian cancer cell proliferation and apoptosis as a potential therapeutic agent. Results T0901317 treatment resulted in a significant (P <0.001) inhibition of cell proliferation in a time- and dose-dependent manner in CaOV3, SKOV3 and A2780 cells. Western blot analysis demonstrated an induction of p21 and p27 with a concominant reduction in phospho-RB protein levels. Cell cycle analysis demonstrated a significant (P <0.001) arrest in the G1 cell cycle phase. Significant induction of Caspase-3 and BAX gene expression occurred with treatment. Induction of apoptosis was confirmed by significant (P < 0.001) elevation of caspase activity on FACS analysis, caspase-glo assay, BAX protein induction and decreased caspase 3 precursor protein expression on Western blot analysis. LXR α/β knockdown experiments did not reverse the anti-proliferative and cytotoxic effects of T0901317. Conclusions The LXR agonist, T0901317, significantly suppresses cell proliferation and induces programmed cell death in a dose- and time-dependent manner. Our results indicate that T0901317 induces its anti-proliferative and cytotoxic effects via an LXR-independent mechanism.

The G protein–coupled receptor GPR30 binds 17β-estradiol (E2) yet differs from classic estrogen receptors (ERα and ERβ). GPR30 can mediate E2-induced nongenomic signaling, but its role in ERα-positive breast cancer remains unclear. Gene expression microarray data from five cohorts comprising 1,250 breast carcinomas showed an association between increased GPR30 expression and ERα-positive status. We therefore examined GPR30 in estrogenic activities in ER-positive MCF-7 breast cancer cells using G-1 and diethylstilbestrol (DES), ligands that selectively activate GPR30 and ER, respectively, and small interfering RNAs. In expression studies, E2 and DES, but not G-1, transiently downregulated both ER and GPR30, indicating that this was ER mediated. In Ca2+ mobilization studies, GPR30, but not ERα, mediated E2-induced Ca2+ responses because E2, 4-hydroxytamoxifen (activates GPR30), and G-1, but not DES, elicited cytosolic Ca2+ increases not only in MCF-7 cells but also in ER-negative SKBr3 cells. Additionally, in MCF-7 cells, GPR30 depletion blocked E2-induced and G-1–induced Ca2+ mobilization, but ERα depletion did not. Interestingly, GPR30-coupled Ca2+ responses were sustained and inositol triphosphate receptor mediated in ER-positive MCF-7 cells but transitory and ryanodine receptor mediated in ER-negative SKBr3 cells. Proliferation studies involving GPR30 depletion indicated that the role of GPR30 was to promote SKBr3 cell growth but reduce MCF-7 cell growth. Supporting this, G-1 profoundly inhibited MCF-7 cell growth, potentially via p53 and p21 induction. Further, flow cytometry showed that G-1 blocked MCF-7 cell cycle progression at the G1 phase. Thus, GPR30 antagonizes growth of ERα-positive breast cancer and may represent a new target to combat this disease. Cancer Res; 70(3); 1184–94

In macrophages, peroxisome proliferator-activated receptor gamma (PPARgamma) has been shown to be important for differentiation, and it serves as a negative regulator of activation. Major trauma/injury causes a dramatic host response that disrupts cellular immune homeostasis and initiates an inflammatory cascade that predisposes the injured host to subsequent infections. In prior studies using a murine trauma model consisting of femur fracture and hemorrhage, splenic macrophages from traumatized mice had significantly enhanced LPS-induced cyclooxygenase enzyme (subtype 2) and iNOS production as well as elevated levels of inflammatory cytokines at 1 week after injury compared with uninjured controls. These up-regulated cellular responses corresponded to increased mortality when animals were challenged with LPS or Candida. In the current study, we used the injury model to determine the effect of treatment of injured mice with the endogenous PPARgamma ligand 15-deoxy-Delta(12-, 14)-PGJ2 (15d-PGJ2). It was found that in vivo 15d-PGJ2 treatment significantly reduced the levels of inflammatory mediators produced by splenic macrophages 7 days after injury. The mechanism of inhibition is dependent on PPARgamma because concomitant treatment of animals with the PPARgamma antagonist GW9662 reversed the inhibitory effect of 15d-PGJ2. Endogenous PPARgamma modulated activation of LPS-induced p38 mitogen-activated protein kinase. Furthermore, treatment of injured mice with 15d-PGJ2 conferred a significant survival advantage after infectious challenge induced by cecal ligation and puncture. Thus, this PPARgamma ligands significantly attenuate the postinjury inflammatory response and improve survival after infectious challenge.

Acquired resistance to long-term antihormonal therapy in breast cancer evolves through two Phases over 5 years. Phase I develops within a year, and tumor growth occurs with either estradiol or tamoxifen. However, Phase II resistance develops after 5 years of therapy. Tamoxifen continues to stimulate tumor growth, however the resistant tumor cell evolves so that estradiol is no longer a survival signal, but instead induces apoptosis. This is the basis for the therapeutic use of estrogen to treat breast cancer. Unfortunately only 30% of tumors respond, so laboratory studies were designed to seek mechanisms to enhance apoptotic responses. We have compared the actions of estradiol in MCF-7 variants that are invulnerable to estradiol-induced apoptosis (wild-type WS8), rapidly induce apoptosis (5C) or exhibit delayed apoptosis (2A). Using Agilent 4×44k human oligonucleotide microarrays, gene expression profiling of these cell lines treated with and without E2 was conducted over a 96 hour time course. Additionally, a long-term 9 day time course of 2A cells was conducted to capture its delayed apoptotic response. A statistically rigorous method was developed to analyze the time course expression profiles in which the area under the curve (AUC) was computed for all genes and compared between cell lines. Focusing on apoptotic genes, caspase-4 was the earliest caspase significantly induced in the 5C cells from 24-96 h, and in the 2A cells from 6-9 days. Caspase-4 is specifically associated with the accumulation of unfolded proteins and protein aggregation in endoplasmic reticulum stress-induced apoptosis. Consistent with this mechanism of apoptosis, the observed gene expression profiles indicated a lack of induction in both resistant cell lines compared to wild-type cells of genes involved in protein folding such as chaperones (e.g. HSPA13, HSP70 member 13), co-chaperones (e.g. AHSA1, activator of HSP90 protein ATPase homolog 1) and chaperonins (HSP60/HPSD1; TCP1, T-complex 1). Additionally, the pro-apoptotic factor Bim, which is also associated with endoplasmic reticulum stress, was induced in both resistant cell lines compared to wild-type cells. Caspase-4 induction was confirmed by quantitative PCR and immunoblotting. The specific caspase-4 inhibitor Z-LEVD-FMK not only reduced cleaved caspase 4, it also completely blocked PARP cleavage, and estradiol-induced apoptosis changes in morphology and growth. Based on these data, a strategy to sensitize cells to estradiol-induced apoptosis could be applied to improve clinical response rates in Phase II acquired resistance to antihormone therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1279.