Predictors of success in establishing orthotopic patient-derived xenograft models of triple negative breast cancer.

Patient-derived xenograft (PDX) models of breast cancer are an effective discovery platform and tool for preclinical pharmacologic testing and biomarker identification. We established orthotopic PDX models of triple negative breast cancer (TNBC) from the primary breast tumors of patients prior to and following neoadjuvant chemotherapy (NACT) while they were enrolled in the ARTEMIS trial (NCT02276443). Serial biopsies were obtained from patients prior to treatment (pre-NACT), from poorly responsive disease after four cycles of Adriamycin and cyclophosphamide (AC, mid-NACT), and in cases of AC-resistance, after a 3-month course of different experimental therapies and/or additional chemotherapy (post-NACT). Our study cohort includes a total of 269 fine needle aspirates (FNAs) from 217 women, generating a total of 62 PDX models (overall success-rate = 23%). Success of PDX engraftment was generally higher from those cancers that proved to be treatment-resistant, whether poorly responsive to AC as determined by ultrasound measurements mid-NACT (p = 0.063), RCB II/III status after NACT (p = 0.046), or metastatic relapse within 2 years of surgery (p = 0.008). TNBC molecular subtype determined from gene expression microarrays of pre-NACT tumors revealed no significant association with PDX engraftment rate (p = 0.877). Finally, we developed a statistical model predictive of PDX engraftment using percent Ki67 positive cells in the patient's diagnostic biopsy, positive lymph node status at diagnosis, and low volumetric reduction of the patient's tumor following AC treatment. This novel bank of 62 PDX models of TNBC provides a valuable resource for biomarker discovery and preclinical therapeutic trials aimed at improving neoadjuvant response rates for patients with TNBC.

Assessment of stained direct cytology smears of breast cancer for whole transcriptome and targeted messenger RNA sequencing.

BACKGROUND: Rather than surgical resection, cytologic specimens are often used as first-line clinical diagnostic procedures due to higher safety, speed, and cost-effectiveness. Archival diagnostic cytology slides containing cancer can be equivalent to tissue biopsies for DNA mutation testing, but the accuracy of transcriptomic profiling by RNA sequencing (RNA-seq) is less understood. METHODS: This study compares the results from whole transcriptome RNA-seq and a targeted RNA-seq assay of stained cytology smears (CS) versus matched tumor tissue samples preserved fresh-frozen (FF) and processed as formalin-fixed paraffin-embedded (FFPE) sections. Cellular cytology scrapes from all 11 breast cancers were fixed and stained using three common protocols: Carnoy's (CS_C) or 95% ethanol (CS_E) fixation and then Papanicolaou stain or air-dried then methanol fixation and DiffQuik stain (CS_DQ). Agreement between samples was assessed using Lin's concordance correlation coefficient. RESULTS: Library yield for CS_DQ was too low, therefore it was not sequenced. The distributions of concordance correlation coefficient of gene expression levels in comparison to FF were comparable between CS_C and CS_E, but expression of genes enriched in stroma was lower in cytosmear samples than in FF or FFPE. Six signatures showed similar concordance to FF for all methods and two were slightly worse in CS_C and CS_E. Genomic signatures were highly concordant using targeted RNA-seq. The allele fraction of selected mutations calculated on cytosmear specimens was highly correlated with FF tissues using both RNA-seq methods. CONCLUSION: RNA can be reliably extracted from cytology smears and is suitable for transcriptome profiling or mutation detection, except for signatures of tumor stroma.

Molecular testing opportunities on cytology effusion specimens: the pre-analytic effects of various body fluid cytology preparation methods on RNA extraction quality and targeted sequencing.

INTRODUCTION: RNA sequencing (RNAseq) analysis is emerging as a clinical research or diagnostic approach for cytologic samples, but there is need for formal comparison of different sample preparation methods in the cytology laboratory to identify which pre-analytic methods could provide alternatives to formalin-fixed paraffin-embedded (FFPE) sections. MATERIALS AND METHODS: We prepared 13 malignant effusions (metastatic estrogen receptor-positive breast cancer) in the cytology laboratory using 6 routine cytologic methods: FFPE cell block, Carnoy's solution, 95% ethanol (EtOH), air-dried and Diff-Quik, ThinPrep, and SurePath preparations. Measurements of RNA quality, expression of 2 multigene expression signatures, molecular subtype, and 4 common activating mutation sites in each preparation were compared with fresh frozen (FF) cell pellet in RNA preservative using distribution of fragment length and concordance correlation coefficient (CCC). RESULTS: The fraction of RNA fragments measuring 200 bases or more (DV200) were 24% higher from cytospins fixed in Carnoy's solution or 95% EtOH than DV200 from FFPE cell blocks. SurePath samples failed RNAseq quality control. There was high concordance of gene expression measurements with FF samples using cytospins fixed in Carnoy's solution, 95% EtOH, Diff-Quik (CCC = 0.829, 0.812, 0.760, respectively), or ThinPrep (CCC = 0.736), but lower using FFPE cell block (CCC = 0.564). The proportion of mutant transcripts was concordant between FF and any cytologic preparation methods. CONCLUSIONS: Cytospin preparations fixed with Carnoy's or 95% ETOH then Papanicolaou stained produced RNAseq results that were equivalent to FF samples and superior to FFPE cell block sections.

Intra- and Interlaboratory Reproducibility of the Sensitivity to Endocrine Therapy Assay for Stage II/III Breast Cancer.

BACKGROUND: The sensitivity to endocrine therapy assay (SET2,3) predicts treatment outcomes in Stage II-III breast cancer. SET2,3 measures transcription related to estrogen and progesterone receptors (SETER/PR index) and the molecular subtype (RNA4: ESR1, PGR, ERBB2, AURKA) from formalin-fixed paraffin-embedded (FFPE) tissue sections. METHODS: We designed a nested study across 3 pathology laboratories, each testing 60 breast cancers twice in controlled batches. Laboratories macrodissected and directly homogenized the unstained FFPE tumor sections, then performed the QuantiGene Plex bead-based hybridization assay. SET2,3 was calculated centrally using predefined statistical R-scripts and applying pre-defined cutpoints. Concordance correlation coefficient (CCC) was calculated from continuous measurements and Kappa statistic from categorical results. A mixed-effects model estimated contributions to bias (fixed effects) and variance (random effects) from the replicated design. RESULTS: Intralaboratory (CCC 0.96-0.99) and interlaboratory (CCC 0.98-0.99) SET2,3 results were concordant, with rates of agreement for high/low categorization within (Kappa 0.83-0.93) and between laboratories (Kappa 0.87-0.88). The relative contributions to overall variance of SET2,3 measurements were 96.90% from biological differences between cancers, 0.67% from interlaboratory variability, and 2.44% from residual causes including intralaboratory replicates. Similar results were obtained with SETER/PR, the baseline prognostic index calculated using pathological or clinical tumor and nodal staging information, and the 4 individual genes (ESR1, PGR, ERBB2, and AURKA). CONCLUSION: Intra- and interpathology laboratory measurements of SET2,3 and its components were highly reproducible when tested from FFPE tumor sections.

Targeted RNAseq assay incorporating unique molecular identifiers for improved quantification of gene expression signatures and transcribed mutation fraction in fixed tumor samples.

BACKGROUND: Our objective was to assess whether modifications to a customized targeted RNA sequencing (RNAseq) assay to include unique molecular identifiers (UMIs) that collapse read counts to their source mRNA counts would improve quantification of transcripts from formalin-fixed paraffin-embedded (FFPE) tumor tissue samples. The assay (SET4) includes signatures that measure hormone receptor and PI3-kinase related transcriptional activity (SETER/PR and PI3Kges), and measures expression of selected activating point mutations and key breast cancer genes. METHODS: Modifications included steps to introduce eight nucleotides-long UMIs during reverse transcription (RT) in bulk solution, followed by polymerase chain reaction (PCR) of labeled cDNA in droplets, with optimization of the polymerase enzyme and reaction conditions. We used Lin's concordance correlation coefficient (CCC) to measure concordance, including precision (Rho) and accuracy (Bias), and nonparametric tests (Wilcoxon, Levene's) to compare the modified (NEW) SET4 assay to the original (OLD) SET4 assay and to whole transcriptome RNAseq using RNA from matched fresh frozen (FF) and FFPE samples from 12 primary breast cancers. RESULTS: The modified (NEW) SET4 assay measured single transcripts (p< 0.001) and SETER/PR (p=0.002) more reproducibly in technical replicates from FFPE samples. The modified SET4 assay was more precise for measuring single transcripts (Rho 0.966 vs 0.888, p< 0.01) but not multigene expression signatures SETER/PR (Rho 0.985 vs 0.968) or PI3Kges (Rho 0.985 vs 0.946) in FFPE, compared to FF samples. It was also more precise than wtRNAseq of FFPE for measuring transcripts (Rho 0.986 vs 0.934, p< 0.001) and SETER/PR (Rho 0.993 vs 0.915, p=0.004), but not PI3Kges (Rho 0.988 vs 0.945, p=0.051). Accuracy (Bias) was comparable between protocols. Two samples carried a PIK3CA mutation, and measurements of transcribed mutant allele fraction was similar in FF and FFPE samples and appeared more precise with the modified SET4 assay. Amplification efficiency (reads per UMI) was consistent in FF and FFPE samples, and close to the theoretically expected value, when the library size exceeded 400,000 aligned reads. CONCLUSIONS: Modifications to the targeted RNAseq protocol for SET4 assay significantly increased the precision of UMI-based and reads-based measurements of individual transcripts, multi-gene signatures, and mutant transcript fraction, particularly with FFPE samples.

Technical Validity of a Customized Assay of Sensitivity to Endocrine Therapy Using Sections from Fixed Breast Cancer Tissue.

BACKGROUND: We translated a multigene expression index to predict sensitivity to endocrine therapy for Stage II-III breast cancer (SET2,3) to hybridization-based expression assays of formalin-fixed paraffin-embedded (FFPE) tissue sections. Here we report the technical validity with FFPE samples, including preanalytical and analytical performance. METHODS: We calibrated SET2,3 from microarrays (Affymetrix U133A) of frozen samples to hybridization-based assays of FFPE tissue, using bead-based QuantiGene Plex (QGP) and slide-based NanoString (NS). The following preanalytical and analytical conditions were tested in controlled studies: replicates within and between frozen and fixed samples, age of paraffin blocks, homogenization of fixed sections versus extracted RNA, core biopsy versus surgically resected tumor, technical replicates, precision over 20 weeks, limiting dilution, linear range, and analytical sensitivity. Lin's concordance correlation coefficient (CCC) was used to measure concordance between measurements. RESULTS: SET2,3 index was calibrated to use with QGP (CCC 0.94) and NS (CCC 0.93) technical platforms, and was validated in two cohorts of older fixed samples using QGP (CCC 0.72, 0.85) and NS (CCC 0.78, 0.78). QGP assay was concordant using direct homogenization of fixed sections versus purified RNA (CCC 0.97) and between core and surgical sample types (CCC 0.90), with 100% accuracy in technical replicates, 1-9% coefficient of variation over 20 weekly tests, linear range 3.0-11.5 (log2 counts), and analytical sensitivity ≥2.0 (log2 counts). CONCLUSIONS: Measurement of the novel SET2,3 assay was technically valid from fixed tumor sections of biopsy or resection samples using simple, inexpensive, hybridization methods, without the need for RNA purification.

The impact of RNA extraction method on accurate RNA sequencing from formalin-fixed paraffin-embedded tissues.

BACKGROUND: Utilization of RNA sequencing methods to measure gene expression from archival formalin-fixed paraffin-embedded (FFPE) tumor samples in translational research and clinical trials requires reliable interpretation of the impact of pre-analytical variables on the data obtained, particularly the methods used to preserve samples and to purify RNA. METHODS: Matched tissue samples from 12 breast cancers were fresh frozen (FF) and preserved in RNAlater or fixed in formalin and processed as FFPE tissue. Total RNA was extracted and purified from FF samples using the Qiagen RNeasy kit, and in duplicate from FFPE tissue sections using three different kits (Norgen, Qiagen and Roche). All RNA samples underwent whole transcriptome RNA sequencing (wtRNAseq) and targeted RNA sequencing for 31 transcripts included in a signature of sensitivity to endocrine therapy. We assessed the effect of RNA extraction kit on the reliability of gene expression levels using linear mixed-effects model analysis, concordance correlation coefficient (CCC) and differential analysis. All protein-coding genes in the wtRNAseq and three gene expression signatures for breast cancer were assessed for concordance. RESULTS: Despite variable quality of the RNA extracted from FFPE samples by different kits, all had similar concordance of overall gene expression from wtRNAseq between matched FF and FFPE samples (median CCC 0.63-0.66) and between technical replicates (median expression difference 0.13-0.22). More than half of genes were differentially expressed between FF and FFPE, but with low fold change (median |LFC| 0.31-0.34). Two out of three breast cancer signatures studied were highly robust in all samples using any kit, whereas the third signature was similarly discordant irrespective of the kit used. The targeted RNAseq assay was concordant between FFPE and FF samples using any of the kits (CCC 0.91-0.96). CONCLUSIONS: The selection of kit to purify RNA from FFPE did not influence the overall quality of results from wtRNAseq, thus variable reproducibility of gene signatures probably relates to the reliability of individual gene selected and possibly to the algorithm. Targeted RNAseq showed promising performance for clinical deployment of quantitative assays in breast cancer from FFPE samples, although numerical scores were not identical to those from wtRNAseq and would require calibration.

Intrinsically Disordered SRC-3/AIB1 Protein Undergoes Homeostatic Nuclear Extrusion by Nuclear Budding While Ectopic Expression Induces Nucleophagy.

SRC-3/AIB1 (Amplified in Breast Cancer-1) is a nuclear receptor coactivator for the estrogen receptor in breast cancer cells. It is also an intrinsically disordered protein when not engaged with transcriptional binding partners and degraded upon transcriptional coactivation. Given the amplified expression of SRC-3 in breast cancers, the objective of this study was to determine how increasing SRC-3 protein levels are regulated in MCF-7 breast cancer cells. We found that endogenous SRC-3 was expelled from the nucleus in vesicle-like spheres under normal growth conditions suggesting that this form of nuclear exclusion of SRC-3 is a homeostatic mechanism for regulating nuclear SRC-3 protein. Only SRC-3 not associated with CREB-binding protein (CBP) was extruded from the nucleus. We found that overexpression in MCF-7 cells results in aneuploid senescence and cell death with frequent formation of nuclear aggregates which were consistently juxtaposed to perinuclear microtubules. Transfected SRC-3 was SUMOylated and caused redistribution of nuclear promyelocytic leukemia (PML) bodies and perturbation of the nuclear membrane lamin B1, hallmarks of nucleophagy. Increased SRC-3 protein-induced autophagy and resulted in SUMO-1 localization to the nuclear membrane and formation of protrusions variously containing SRC-3 and chromatin. Aspects of SRC-3 overexpression and toxicity were recapitulated following treatment with clinically relevant agents that stabilize SRC-3 in breast cancer cells. We conclude that amplified SRC-3 levels have major impacts on nuclear protein quality control pathways and may mark cancer cells for sensitivity to protein stabilizing therapeutics.

SETER/PR: a robust 18-gene predictor for sensitivity to endocrine therapy for metastatic breast cancer.

There is a clinical need to predict sensitivity of metastatic hormone receptor-positive and HER2-negative (HR+/HER2-) breast cancer to endocrine therapy, and targeted RNA sequencing (RNAseq) offers diagnostic potential to measure both transcriptional activity and functional mutation. We developed the SETER/PR index to measure gene expression microarray probe sets that were correlated with hormone receptors (ESR1 and PGR) and robust to preanalytical and analytical influences. We tested SETER/PR index in biopsies of metastastic HR+/HER2- breast cancer against the treatment outcomes in 140 patients. Then we customized the SETER/PR assay to measure 18 informative, 10 reference transcripts, and sequence the ligand-binding domain (LBD) of ESR1 using droplet-based targeted RNAseq, and tested that in residual RNA from 53 patients. Higher SETER/PR index in metastatic samples predicted longer PFS and OS when patients received endocrine therapy as next treatment, even after adjustment for clinical-pathologic risk factors (PFS: HR 0.534, 95% CI 0.299 to 0.955, p = 0.035; OS: HR 0.315, 95% CI 0.157 to 0.631, p = 0.001). Mutated ESR1 LBD was detected in 8/53 (15%) of metastases, involving 1-98% of ESR1 transcripts (all had high SETER/PR index). A signature based on probe sets with good preanalytical and analytical performance facilitated our customization of an accurate targeted RNAseq assay to measure both phenotype and genotype of ER-related transcription. Elevated SETER/PR was associated with prolonged sensitivity to endocrine therapy in patients with metastatic HR+/HER2- breast cancer, especially in the absence of mutated ESR1 transcript.

Resistance to neoadjuvant chemotherapy in triple-negative breast cancer mediated by a reversible drug-tolerant state.

Eradicating triple-negative breast cancer (TNBC) resistant to neoadjuvant chemotherapy (NACT) is a critical unmet clinical need. In this study, patient-derived xenograft (PDX) models of treatment-naïve TNBC and serial biopsies from TNBC patients undergoing NACT were used to elucidate mechanisms of chemoresistance in the neoadjuvant setting. Barcode-mediated clonal tracking and genomic sequencing of PDX tumors revealed that residual tumors remaining after treatment with standard frontline chemotherapies, doxorubicin (Adriamycin) combined with cyclophosphamide (AC), maintained the subclonal architecture of untreated tumors, yet their transcriptomes, proteomes, and histologic features were distinct from those of untreated tumors. Once treatment was halted, residual tumors gave rise to AC-sensitive tumors with similar transcriptomes, proteomes, and histological features to those of untreated tumors. Together, these results demonstrated that tumors can adopt a reversible drug-tolerant state that does not involve clonal selection as an AC resistance mechanism. Serial biopsies obtained from patients with TNBC undergoing NACT revealed similar histologic changes and maintenance of stable subclonal architecture, demonstrating that AC-treated PDXs capture molecular features characteristic of human TNBC chemoresistance. Last, pharmacologic inhibition of oxidative phosphorylation using an inhibitor currently in phase 1 clinical development delayed residual tumor regrowth. Thus, AC resistance in treatment-naïve TNBC can be mediated by nonselective mechanisms that confer a reversible chemotherapy-tolerant state with targetable vulnerabilities.

cIAP2 Is an Independent Signaling and Survival Factor during Mammary Lactational Involution and Tumorigenesis.

Cellular inhibitor of apoptosis proteins-1 and -2 (cIAP1/2) are integral to regulation of apoptosis and signaling by the tumor necrosis factor (TNF) and related family of receptors. The expression of cIAP2 in tissues is typically low and considered functionally redundant with cIAP1, however cIAP2 can be activated by a variety of cellular stresses. Members of the TNFR family and their ligands have essential roles in mammary gland biology. We have found that cIAP2-/- virgin mammary glands have reduced ductal branching and delayed lobuloalveogenesis in early pregnancy. Post-lactational involution involves two phases where the first phase is reversible and is mediated, in part, by TNFR family ligands. In cIAP2-/- mice mammary glands appeared engorged at mid-lactation accompanied by enhanced autophagic flux and decreased cIAP1 protein expression. Severely stretched myoepithelium was associated with BIM-EL expression and other indicators of anoikis. Within 24 h after forced or natural weaning, cIAP2-/- glands had nearly completed involution. The TNF-related weak inducer of apoptosis (Tweak) which results in degradation of cIAP1 through its receptor, Fn14, began to increase in late lactation and was significantly increased in cIAP2-/- relative to WT mice by 12 h post weaning accompanied by decreased cIAP1 protein expression. Carcinogen/progesterone-induced mammary tumorigenesis was significantly delayed in cIAP2-/- mice and tumors contained high numbers of apoptotic cells. We conclude that cIAP2 has a critical role in the mammary gland wherein it prevents rapid involution induced by milk stasis-induced stress associated with Tweak activation and contributes to the survival of mammary tumor cells.

Predictors of Chemosensitivity in Triple Negative Breast Cancer: An Integrated Genomic Analysis.

BACKGROUND: Triple negative breast cancer (TNBC) is a highly heterogeneous and aggressive disease, and although no effective targeted therapies are available to date, about one-third of patients with TNBC achieve pathologic complete response (pCR) from standard-of-care anthracycline/taxane (ACT) chemotherapy. The heterogeneity of these tumors, however, has hindered the discovery of effective biomarkers to identify such patients. METHODS AND FINDINGS: We performed whole exome sequencing on 29 TNBC cases from the MD Anderson Cancer Center (MDACC) selected because they had either pCR (n = 18) or extensive residual disease (n = 11) after neoadjuvant chemotherapy, with cases from The Cancer Genome Atlas (TCGA; n = 144) and METABRIC (n = 278) cohorts serving as validation cohorts. Our analysis revealed that mutations in the AR- and FOXA1-regulated networks, in which BRCA1 plays a key role, are associated with significantly higher sensitivity to ACT chemotherapy in the MDACC cohort (pCR rate of 94.1% compared to 16.6% in tumors without mutations in AR/FOXA1 pathway, adjusted p = 0.02) and significantly better survival outcome in the TCGA TNBC cohort (log-rank test, p = 0.05). Combined analysis of DNA sequencing, DNA methylation, and RNA sequencing identified tumors of a distinct BRCA-deficient (BRCA-D) TNBC subtype characterized by low levels of wild-type BRCA1/2 expression. Patients with functionally BRCA-D tumors had significantly better survival with standard-of-care chemotherapy than patients whose tumors were not BRCA-D (log-rank test, p = 0.021), and they had significantly higher mutation burden (p < 0.001) and presented clonal neoantigens that were associated with increased immune cell activity. A transcriptional signature of BRCA-D TNBC tumors was independently validated to be significantly associated with improved survival in the METABRIC dataset (log-rank test, p = 0.009). As a retrospective study, limitations include the small size and potential selection bias in the discovery cohort. CONCLUSIONS: The comprehensive molecular analysis presented in this study directly links BRCA deficiency with increased clonal mutation burden and significantly enhanced chemosensitivity in TNBC and suggests that functional RNA-based BRCA deficiency needs to be further examined in TNBC.

Persistent Activation of NF-κB in BRCA1-Deficient Mammary Progenitors Drives Aberrant Proliferation and Accumulation of DNA Damage.

Human BRCA1 mutation carriers and BRCA1-deficient mouse mammary glands contain an abnormal population of mammary luminal progenitors that can form 3D colonies in a hormone-independent manner. The intrinsic cellular regulatory defect in these presumptive breast cancer precursors is not known. We have discovered that nuclear factor kappaB (NF-κB) (p52/RelB) is persistently activated in a subset of BRCA1-deficient mammary luminal progenitors. Hormone-independent luminal progenitor colony formation required NF-κB, ataxia telangiectasia-mutated (ATM), and the inhibitor of kappaB kinase, IKKα. Progesterone (P4)-stimulated proliferation resulted in a marked enhancement of DNA damage foci in Brca1(-/-) mouse mammary. In vivo, NF-κB inhibition prevented recovery of Brca1(-/-) hormone-independent colony-forming cells. The majority of human BRCA1(mut/+) mammary glands showed marked lobular expression of nuclear NF-κB. We conclude that the aberrant proliferative capacity of Brca1(-/-) luminal progenitor cells is linked to the replication-associated DNA damage response, where proliferation of mammary progenitors is perpetuated by damage-induced, autologous NF-κB signaling.

Voices of Alzheimer Caregivers on Positive Aspects of Caregiving.

PURPOSE: To discover positive gains as constructed by family caregivers of relatives with Alzheimer's disease. DESIGN AND METHODS: Fifty-seven Hong Kong Chinese primary caregivers provided 669 diary recordings over an 8-week period, describing daily events and experiences in which positive gains were achieved. The diaries were analyzed independently by two researchers using thematic analysis. RESULTS: Ten themes related to positive gains were identified: (a) insights about dementia and acceptance of the condition, (b) a sense of purpose and commitment to the caregiving role, (c) feelings of gratification when the care-recipient (CR) was functioning relatively well, (d) mastering skills to handle the CR, (e) increased patience and tolerance, (f) cultivating positive meanings and humor amidst difficult circumstances, (g) letting go of things, such as when the CR's qualities had been lost or personal agenda had become unrealistic, (h) developing a closer relationship with the CR, (i) finding support, and (j) feeling useful helping other caregivers. IMPLICATIONS: In addition to treating negative outcomes such as depression, practitioners and researchers should, identify means to promote positive gains. Strategies mentioned in the caregivers' diaries, such as reframing stressful situations in a more positive light, may provide input into the design of such interventions.

Preferential estrogen receptor ß ligands reduce Bcl-2 expression in hormone-resistant breast cancer cells to increase autophagy.

Acquired resistance to selective estrogen receptor (ER) modulators (SERM) and downregulators (SERD) is a significant clinical problem in the treatment of estrogen (E2) receptor-positive (ER(+)) breast cancers. There are two ER subtypes, ERα and ERß, which promote and inhibit breast cancer cell proliferation, respectively. Although ER(+) breast cancers typically express a high ratio of ERα to ERß, the acquisition of SERM resistance in vitro and in vivo is associated with increased relative expression of the ERß. On some gene enhancers, ERß has been shown to function in opposition to the ERα in the presence of E2. Here, we demonstrate that two different ERß agonists, WAY-20070 and a novel "A-CD" estrogen called L17, produce a marked reduction in G(2)-M phase correlated with effects on cyclin D1 and cyclin E expression in a SERM/SERD-resistant breast cancer cell line. ERß agonists recruited both the ERα and ERß to the Bcl-2 E2-response element strongly reducing Bcl-2 mRNA and protein in an ERß-dependent manner. L17 recruited RIP140 to the Bcl-2 promoter in cells overexpressing ERß. Exposure to the ERß ligands also resulted in increased processing of LC3-I to LC3-II, indicative of enhanced autophagic flux. The coaddition of ERß agonist and the autophagy inhibitor chloroquine resulted in a significant accumulation of sub-G1 DNA which was completely prevented by the addition of the caspase inhibitor Z-VAD-FMK. We propose that combined therapies with an ERß agonist and an inhibitor of autophagy may provide the basis for a novel approach to the treatment of SERM/SERD-resistant breast cancers.

Benefit-finding intervention for Alzheimer caregivers: conceptual framework, implementation issues, and preliminary efficacy.

PURPOSE: To describe an intervention promoting benefit-finding in Alzheimer caregivers, to discuss key issues in implementation and ways to resolve them, and to examine whether the intervention reduced burden and depression in a small randomized trial. DESIGN AND METHODS: Twenty-five caregivers were randomized into benefit-finding and psychoeducation groups. Both groups had eight weekly sessions. Outcome measures including role overload, Zarit Burden Interview, and Hamilton depression scale were collected at baseline and after treatment. Results were analyzed using analysis of covariance. Additionally, the challenges of implementing such interventions, some of which related to cultural issues, were analyzed qualitatively. RESULTS: Controlling for pretest, the benefit-finding group had lower depression than the psychoeducation group at post-test, despite the fact that some caregivers found benefit-finding challenging. The two groups did not differ on overload and burden. However, within-group analysis suggested that both groups showed significant reductions in overload from pretest to post-test. In addition, we discussed participants' difficulties in grasping the technique of thought modification for benefit-finding, recording such exercises at home, and sharing their thoughts and experiences in groups. We described measures undertaken in the main trial to overcome these issues. IMPLICATIONS: Cognitive approaches focusing on benefit-finding are feasible among Chinese caregivers, with preliminary evidence suggesting an effect on alleviating depression.

NF-κB-dependent role for cold-inducible RNA binding protein in regulating interleukin 1ß.

The cold inducible RNA binding protein (CIRBP) responds to a wide array of cellular stresses, including short wavelength ultraviolet light (UVC), at the transcriptional and post-translational level. CIRBP can bind the 3'untranslated region of specific transcripts to stabilize them and facilitate their transport to ribosomes for translation. Here we used RNA interference and oligonucleotide microarrays to identify potential downstream targets of CIRBP induced in response to UVC. Twenty eight transcripts were statistically increased in response to UVC and these exhibited a typical UVC response. Only 5 of the 28 UVC-induced transcripts exhibited a CIRBP-dependent pattern of expression. Surprisingly, 3 of the 5 transcripts (IL1B, IL8 and TNFAIP6) encoded proteins important in inflammation with IL-1ß apparently contributing to IL8 and TNFAIP6 expression in an autocrine fashion. UVC-induced IL1B expression could be inhibited by pharmacological inhibition of NFκB suggesting that CIRBP was affecting NF-κB signaling as opposed to IL1B mRNA stability directly. Bacterial lipopolysaccharide (LPS) was used as an activator of NF-κB to further study the potential link between CIRBP and NFκB. Transfection of siRNAs against CIRBP reduced the extent of the LPS-induced phosphorylation of IκBα, NF-κB DNA binding activity and IL-1ß expression. The present work firmly establishes a novel link between CIRBP and NF-κB signaling in response to agents with diverse modes of action. These results have potential implications for disease states associated with inflammation.

cIAP2 represses IKKα/ß-mediated activation of MDM2 to prevent p53 degradation.

Cellular inhibitor of apoptosis proteins (cIAP1 and cIAP2) function to prevent apoptosis and are often overexpressed in various cancers. However, mutations in cIAP1/2 can activate the alternative NFκB pathway through IκBα-kinase-α (IKKα) and are associated with hematopoetic malignancies. In the current study, we found that knockdown of cIAP2 in human mammary epithelial cells resulted in activation of MDM2 through increased SUMOylation and profound reduction of the pool of MDM2 not phosphorylated at Ser166. cIAP2 siRNA markedly decreased p53 levels, which were rescued by addition of the MDM2 inhibitor, Nutlin3a. An IAP antagonist, which induces cIAP degradation, transiently increased MDM2 mRNA. Simultaneous transfection of siRNA for cIAP2 and IKKα reduced MDM2 protein, while expression of a kinase-dead IKKß strongly increased non-Ser166 P-MDM2. Inhibition of either IKKα or -ß partially rescued p53 levels, while concomitant IKKα/ß inhibition fully rescued p53 after cIAP2 knockdown. Surprisingly, IKKα knockdown alone increased SUMO-MDM2, suggesting that in the absence of activation, IKKα can prevent MDM2 SUMOylation. cIAP2 knockdown disrupted the interaction between the MDM2 SUMO ligase, PIAS1 and IKKα. Partial knockdown of cIAP2 cooperated with (V12) H-ras-transfected mammary epithelial cells to enhance colony formation. In summary, our data identify a novel role for cIAP2 in maintaining wild-type p53 levels by preventing both an NFκB-mediated increase and IKKα/-ß-dependent transcriptional and post-translational modifications of MDM2. Thus, mutations or reductions in cIAP2 could contribute to cancer promotion, in part, through downregulation of p53.

A benefit-finding intervention for family caregivers of persons with Alzheimer disease: study protocol of a randomized controlled trial.

BACKGROUND: Caregivers of relatives with Alzheimer's disease are highly stressed and at risk for physical and psychiatric conditions. Interventions are usually focused on providing caregivers with knowledge of dementia, skills, and/or support, to help them cope with the stress. This model, though true to a certain extent, ignores how caregiver stress is construed in the first place. Besides burden, caregivers also report rewards, uplifts, and gains, such as a sense of purpose and personal growth. Finding benefits through positive reappraisal may offset the effect of caregiving on caregiver outcomes. DESIGN: Two randomized controlled trials are planned. They are essentially the same except that Trial 1 is a cluster trial (that is, randomization based on groups of participants) whereas in Trial 2, randomization is based on individuals. Participants are randomized into three groups - benefit finding, psychoeducation, and simplified psychoeducation. Participants in each group receive a total of approximately 12 hours of training either in group or individually at home. Booster sessions are provided at around 14 months after the initial treatment. The primary outcomes are caregiver stress (subjective burden, role overload, and cortisol), perceived benefits, subjective health, psychological well-being, and depression. The secondary outcomes are caregiver coping, and behavioral problems and functional impairment of the care-recipient. Outcome measures are obtained at baseline, post-treatment (2 months), and 6, 12, 18 and 30 months. DISCUSSION: The emphasis on benefits, rather than losses and difficulties, provides a new dimension to the way interventions for caregivers can be conceptualized and delivered. By focusing on the positive, caregivers may be empowered to sustain caregiving efforts in the long term despite the day-to-day challenges. The two parallel trials will provide an assessment of whether the effectiveness of the intervention depends on the mode of delivery. TRIAL REGISTRATION: Chinese Clinical Trial Registry (http://www.chictr.org/en/) identifier number ChiCTR-TRC-10000881.