Treatment scheduling effects on the evolution of drug resistance in heterogeneous cancer cell populations.

The effect of scheduling of targeted therapy combinations on drug resistance is underexplored in triple-negative breast cancer (TNBC). TNBC constitutes heterogeneous cancer cell populations the composition of which can change dynamically during treatment resulting in the selection of resistant clones with a fitness advantage. We evaluated crizotinib (ALK/MET inhibitor) and navitoclax (ABT-263; Bcl-2/Bcl-xL inhibitor) combinations in a large design consisting of 696 two-cycle sequential and concomitant treatment regimens with varying treatment dose, duration, and drug holiday length over a 26-day period in MDA-MB-231 TNBC cells and found that patterns of resistance depend on the schedule and sequence in which the drugs are given. Further, we tracked the clonal dynamics and mechanisms of resistance using DNA-integrated barcodes and single-cell RNA sequencing. Our study suggests that longer formats of treatment schedules in vitro screening assays are required to understand the effects of resistance and guide more realistically in vivo and clinical studies.

Whole-genome sequencing of phenotypically distinct inflammatory breast cancers reveals similar genomic alterations to non-inflammatory breast cancers.

BACKGROUND: Inflammatory breast cancer (IBC) has a highly invasive and metastatic phenotype. However, little is known about its genetic drivers. To address this, we report the largest cohort of whole-genome sequencing (WGS) of IBC cases. METHODS: We performed WGS of 20 IBC samples and paired normal blood DNA to identify genomic alterations. For comparison, we used 23 matched non-IBC samples from the Cancer Genome Atlas Program (TCGA). We also validated our findings using WGS data from the International Cancer Genome Consortium (ICGC) and the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium. We examined a wide selection of genomic features to search for differences between IBC and conventional breast cancer. These include (i) somatic and germline single-nucleotide variants (SNVs), in both coding and non-coding regions; (ii) the mutational signature and the clonal architecture derived from these SNVs; (iii) copy number and structural variants (CNVs and SVs); and (iv) non-human sequence in the tumors (i.e., exogenous sequences of bacterial origin). RESULTS: Overall, IBC has similar genomic characteristics to non-IBC, including specific alterations, overall mutational load and signature, and tumor heterogeneity. In particular, we observed similar mutation frequencies between IBC and non-IBC, for each gene and most cancer-related pathways. Moreover, we found no exogenous sequences of infectious agents specific to IBC samples. Even though we could not find any strongly statistically distinguishing genomic features between the two groups, we did find some suggestive differences in IBC: (i) The MAST2 gene was more frequently mutated (20% IBC vs. 0% non-IBC). (ii) The TGF ß pathway was more frequently disrupted by germline SNVs (50% vs. 13%). (iii) Different copy number profiles were observed in several genomic regions harboring cancer genes. (iv) Complex SVs were more frequent. (v) The clonal architecture was simpler, suggesting more homogenous tumor-evolutionary lineages. CONCLUSIONS: Whole-genome sequencing of IBC manifests a similar genomic architecture to non-IBC. We found no unique genomic alterations shared in just IBCs; however, subtle genomic differences were observed including germline alterations in TGFß pathway genes and somatic mutations in the MAST2 kinase that could represent potential therapeutic targets.

Multi-Omics Investigation of Innate Navitoclax Resistance in Triple-Negative Breast Cancer Cells.

Cancer cells employ various defense mechanisms against drug-induced cell death. Investigating multi-omics landscapes of cancer cells before and after treatment can reveal resistance mechanisms and inform new therapeutic strategies. We assessed the effects of navitoclax, a BCL2 family inhibitor, on the transcriptome, methylome, chromatin structure, and copy number variations of MDA-MB-231 triple-negative breast cancer (TNBC) cells. Cells were sampled before treatment, at 72 h of exposure, and after 10-day drug-free recovery from treatment. We observed transient alterations in the expression of stress response genes that were accompanied by corresponding changes in chromatin accessibility. Most of these changes returned to baseline after the recovery period. We also detected lasting alterations in methylation states and genome structure that suggest permanent changes in cell population composition. Using single-cell analyses, we identified 2350 genes significantly upregulated in navitoclax-resistant cells and derived an 18-gene navitoclax resistance signature. We assessed the navitoclax-response-predictive function of this signature in four additional TNBC cell lines in vitro and in silico in 619 cell lines treated with 251 different drugs. We observed a drug-specific predictive value in both experiments, suggesting that this signature could help guiding clinical biomarker studies involving navitoclax.

Analysis of Pre- and Posttreatment Tissues from the SWOG S0800 Trial Reveals an Effect of Neoadjuvant Chemotherapy on the Breast Cancer Genome.

PURPOSE: We performed whole-exome sequencing (WES) of pre- and posttreatment cancer tissues to assess the somatic mutation landscape of tumors before and after neoadjuvant taxane and anthracycline chemotherapy with or without bevacizumab. EXPERIMENTAL DESIGN: Twenty-nine pretreatment biopsies from the SWOG S0800 trial were subjected to WES to identify mutational patterns associated with response to neoadjuvant chemotherapy. Nine matching samples with residual cancer after therapy were also analyzed to assess changes in mutational patterns in response to therapy. RESULTS: In pretreatment samples, a higher proportion of mutation signature 3, a BRCA-mediated DNA repair deficiency mutational signature, was associated with higher rate of pathologic complete response (pCR; median signature weight 24%, range 0%-38% in pCR vs. median weight 0%, range 0%-19% in residual disease, Wilcoxon rank sum, Bonferroni P = 0.007). We found no biological pathway level mutations associated with pCR or enriched in posttreatment samples. We observed statistically significant enrichment of high functional impact mutations in the "E2F targets" and "G2-M checkpoint" pathways in residual cancer samples implicating these pathways in resistance to therapy and a significant depletion of mutations in the "myogenesis pathway" suggesting the cells harboring these variants were effectively eradicated by therapy. CONCLUSIONS: These results suggest that genomic disturbances in BRCA-related DNA repair mechanisms, reflected by a dominant mutational signature 3, confer increased chemotherapy sensitivity. Cancers that survive neoadjuvant chemotherapy frequently have alterations in cell-cycle-regulating genes but different genes of the same pathways are affected in different patients.

Identification and Validation of a Novel Biologics Target in Triple Negative Breast Cancer.

The goal of this study was to identify a novel target for antibody-drug conjugate (ADC) development in triple negative breast cancer (TNBC), which has limited treatment options, using gene expression datasets and in vitro siRNA/CRISPR and in vivo functional assays. We analyzed 4467 breast cancers and identified GABRP as top expressed gene in TNBC with low expression in most normal tissues. GABRP protein was localized to cell membrane with broad range of receptors/cell (815-53,714) and expressed by nearly half of breast cancers tissues. GABRP gene knockdown inhibited TNBC cell growth and colony formation in vitro and growth of MDA-MB-468 xenografts in nude mice. Commercially available anti-GABRP antibody (5-100 µg/ml) or de novo generated Fabs (20 µg/ml) inhibited TNBC cell growth in vitro. The same antibody conjugated to mertansine (DM1) also showed significant anticancer activity at nanomolar concentrations. Our results indicate that GABRP is a potential novel therapeutic target for ADC development.

Reliability of Whole-Exome Sequencing for Assessing Intratumor Genetic Heterogeneity.

Multi-region sequencing is used to detect intratumor genetic heterogeneity (ITGH) in tumors. To assess whether genuine ITGH can be distinguished from sequencing artifacts, we performed whole-exome sequencing (WES) on three anatomically distinct regions of the same tumor with technical replicates to estimate technical noise. Somatic variants were detected with three different WES pipelines and subsequently validated by high-depth amplicon sequencing. The cancer-only pipeline was unreliable, with about 69% of the identified somatic variants being false positive. Even with matched normal DNA for which 82% of the somatic variants were detected reliably, only 36%-78% were found consistently in technical replicate pairs. Overall, 34%-80% of the discordant somatic variants, which could be interpreted as ITGH, were found to constitute technical noise. Excluding mutations affecting low-mappability regions or occurring in certain mutational contexts was found to reduce artifacts, yet detection of subclonal mutations by WES in the absence of orthogonal validation remains unreliable.

Increased epigenetic age in normal breast tissue from luminal breast cancer patients.

BACKGROUND: Age is one of the most important risk factors for developing breast cancer. However, age-related changes in normal breast tissue that potentially lead to breast cancer are incompletely understood. Quantifying tissue-level DNA methylation can contribute to understanding these processes. We hypothesized that occurrence of breast cancer should be associated with an acceleration of epigenetic aging in normal breast tissue. RESULTS: Ninety-six normal breast tissue samples were obtained from 88 subjects (breast cancer = 35 subjects/40 samples, unaffected = 53 subjects/53 samples). Normal tissue samples from breast cancer patients were obtained from distant non-tumor sites of primary mastectomy specimens, while samples from unaffected women were obtained from the Komen Tissue Bank (n = 25) and from non-cancer-related breast surgery specimens (n = 28). Patients were further stratified into four cohorts: age < 50 years with and without breast cancer and age ≥ 50 with and without breast cancer. The Illumina HumanMethylation450k BeadChip microarray was used to generate methylation profiles from extracted DNA samples. Data was analyzed using the "Epigenetic Clock," a published biomarker of aging based on a defined set of 353 CpGs in the human genome. The resulting age estimate, DNA methylation age, was related to chronological age and to breast cancer status. The DNAmAge of normal breast tissue was strongly correlated with chronological age (r = 0.712, p < 0.001). Compared to unaffected peers, breast cancer patients exhibited significant age acceleration in their normal breast tissue (p = 0.002). Multivariate analysis revealed that epigenetic age acceleration in the normal breast tissue of subjects with cancer remained significant after adjusting for clinical and demographic variables. Additionally, smoking was found to be positively correlated with epigenetic aging in normal breast tissue (p = 0.012). CONCLUSIONS: Women with luminal breast cancer exhibit significant epigenetic age acceleration in normal adjacent breast tissue, which is consistent with an analogous finding in malignant breast tissue. Smoking is also associated with epigenetic age acceleration in normal breast tissue. Further studies are needed to determine whether epigenetic age acceleration in normal breast tissue is predictive of incident breast cancer and whether this mediates the risk of chronological age on breast cancer risk.

Mitotic read-out genes confer poor outcome in luminal A breast cancer tumors.

Luminal breast tumors have been classified into A and B subgroups, with the luminal A being associated with a more favorable clinical outcome. Unfortunately, luminal A tumors do not have a universally good prognosis. We used transcriptomic analyses using public datasets to evaluate the differential expression between normal breast tissue and breast cancer, focusing on upregulated genes included in cell cycle function. Association of selected genes with relapse free survival (RFS) and overall survival (OS) was performed using the KM Plotter Online Tool (http://www.kmplot.com). Seventy-seven genes were differentially expressed between normal and malignant breast tissue. Only five genes were associated with poor RFS and OS. The mitosis-related genes GTSE1, CDCA3, FAM83D and SMC4 were associated with poor outcome specifically in Luminal A tumors. The combination of FAM83D and CDCA3 for RFS and GTSE1 alone for OS showed the better prediction for clinical outcome. CDCA3 was amplified in 3.4% of the tumors, and FAM83D and SMC4 in 2.3% and 2.2%, respectively. In conclusion, we describe a set of genes that predict detrimental outcome in Luminal A tumors. These genes may have utility for stratification in trials of antimitotic agents or cytotoxic chemotherapy, or as candidates for direct target inhibition.

Intratumor Heterogeneity of Homologous Recombination Deficiency in Primary Breast Cancer.

Purpose: The 3-biomarker homologous recombination deficiency (HRD) assay measures the number of telomeric allelic imbalances, loss of heterozygosity, and large-scale state transitions in tumor DNA and combines these metrics into a single score that reflects DNA repair deficiency. The goal of this study is to assess the consistency of these HRD measures in different biopsies from distinct areas of the same cancer.Experimental Design: HRD scores, BRCA mutation status, and BRCA1 promoter methylation were assessed in 99 samples from 33 surgically resected, stage I-III breast cancers; each cancer was biopsied in three distinct areas. Homologous recombination repair (HR) deficiency was defined as either high HRD score (≥42) or tumor BRCA mutation.Results: Eighty-one biopsies from 32 cancers were analyzed. Tumor BRCA status was available for all samples, HRD scores for 70, and BRCA1 methylation values for 76 samples. The BRCA1/2 mutation and promoter methylation status and HR category showed perfect concordance across all biopsies from the same cancer. All tumors with BRCA1/2 mutations or promoter methylation had high HRD scores, as did 17% (4/24) of the BRCA1/2 wild-type and nonmethylated tumors. The HRD scores were also highly consistent between different biopsies from the same tumor with an intraclass correlation coefficient of 0.977, indicating that only 2.3% of the variance is attributed to within-tumor biopsy-to-biopsy variation.Conclusions: These results indicate that within-tumor spatial heterogeneity for HRD metrics and the technical noise in the assay are small and do not influence HRD scores and HR status. Clin Cancer Res; 23(5); 1193-9. ©2016 AACR.

Systematic Drug Screening Identifies Tractable Targeted Combination Therapies in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) remains an aggressive disease without effective targeted therapies. In this study, we addressed this challenge by testing 128 FDA-approved or investigational drugs as either single agents or in 768 pairwise drug combinations in TNBC cell lines to identify synergistic combinations tractable to clinical translation. Medium-throughput results were scrutinized and extensively analyzed for sensitivity patterns, synergy, anticancer activity, and were validated in low-throughput experiments. Principal component analysis revealed that a fraction of all upregulated or downregulated genes of a particular targeted pathway could partly explain cell sensitivity toward agents targeting that pathway. Combination therapies deemed immediately tractable to translation included ABT-263/crizotinib, ABT-263/paclitaxel, paclitaxel/JQ1, ABT-263/XL-184, and paclitaxel/nutlin-3, all of which exhibited synergistic antiproliferative and apoptotic activity in multiple TNBC backgrounds. Mechanistic investigations of the ABT-263/crizotinib combination offering a potentially rapid path to clinic demonstrated RTK blockade, inhibition of mitogenic signaling, and proapoptotic signal induction in basal and mesenchymal stem-like TNBC. Our findings provide preclinical proof of concept for several combination treatments of TNBC, which offer near-term prospects for clinical translation. Cancer Res; 77(2); 566-78. ©2016 AACR.

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.

miR-34a Silences c-SRC to Attenuate Tumor Growth in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype with no clinically proven biologically targeted treatment options. The molecular heterogeneity of TNBC and lack of high frequency driver mutations other than TP53 have hindered the development of new and effective therapies that significantly improve patient outcomes. miRNAs, global regulators of survival and proliferation pathways important in tumor development and maintenance, are becoming promising therapeutic agents. We performed miRNA-profiling studies in different TNBC subtypes to identify miRNAs that significantly contribute to disease progression. We found that miR-34a was lost in TNBC, specifically within mesenchymal and mesenchymal stem cell-like subtypes, whereas expression of miR-34a targets was significantly enriched. Furthermore, restoration of miR-34a in cell lines representing these subtypes inhibited proliferation and invasion, activated senescence, and promoted sensitivity to dasatinib by targeting the proto-oncogene c-SRC. Notably, SRC depletion in TNBC cell lines phenocopied the effects of miR-34a reintroduction, whereas SRC overexpression rescued the antitumorigenic properties mediated by miR-34a. miR-34a levels also increased when cells were treated with c-SRC inhibitors, suggesting a negative feedback exists between miR-34a and c-SRC. Moreover, miR-34a administration significantly delayed tumor growth of subcutaneously and orthotopically implanted tumors in nude mice, and was accompanied by c-SRC downregulation. Finally, we found that miR-34a and SRC levels were inversely correlated in human tumor specimens. Together, our results demonstrate that miR-34a exerts potent antitumorigenic effects in vitro and in vivo and suggests that miR-34a replacement therapy, which is currently being tested in human clinical trials, represents a promising therapeutic strategy for TNBC.

Characterization of DNA variants in the human kinome in breast cancer.

Kinases play a key role in cancer biology, and serve as potential clinically useful targets for designing cancer therapies. We examined nucleic acid variations in the human kinome and several known cancer-related genes in breast cancer. DNA was extracted from fine needle biopsies of 73 primary breast cancers and 19 metastatic lesions. Targeted sequencing of 518 kinases and 68 additional cancer related genes was performed using the SOLiD sequencing platform. We detected 1561 unique, non-synonymous variants in kinase genes in the 92 cases, and 74 unique variants in 43 kinases that were predicted to have major functional impact on the protein. Three kinase groups--CMGC, STE and TKL--showed greater mutational load in metastatic compared to primary cancer samples, however, after correction for multiple testing the difference was significant only for the TKL group (P = 0.04). We also observed that a higher proportion of histologic grade 1 and 2 cases had high functional impact variants in the SCYL2 gene compared with grade 3 cases. Our findings indicate that individual breast cancers harbor a substantial number of potentially functionally important nucleotide variations in kinase genes, most of which are present in unique combinations and include both somatic and germline functional variants.

Overexpression of ERBB4 JM-a CYT-1 and CYT-2 isoforms in transgenic mice reveals isoform-specific roles in mammary gland development and carcinogenesis.

INTRODUCTION: Human Epidermal Growth Factor Receptor (ERBB4/HER4) belongs to the Epidermal Growth Factor receptor/ERBB family of receptor tyrosine kinases. While ERBB1, ERBB2 and ERBB3 are often overexpressed or activated in breast cancer, and are oncogenic, the role of ERBB4 in breast cancer is uncertain. Some studies suggest a tumor suppressor role of ERBB4, while other reports suggest an oncogenic potential. Alternative splicing of ERBB4 yields four major protein products, these spliced isoforms differ in the extracellular juxtamembrane domain (JM-a versus JM-b) and cytoplasmic domain (CYT-1 versus CYT-2). Two of these isoforms, JM-a CYT-1 and JM-a CYT-2, are expressed in the mammary gland. Failure to account for isoform-specific functions in previous studies may account for conflicting reports on the role of ERBB4 in breast cancer. METHODS: We have produced mouse mammary tumour virus (MMTV) -ERBB4 transgenic mice to evaluate potential developmental and carcinogenic changes associated with full length (FL) JM-a ERBB4 CYT-1 versus ERBB4 CYT-2. Mammary tissue was isolated from transgenic mice and sibling controls at various developmental stages for whole mount analysis, RNA extraction, and immunohistochemistry. To maintain maximal ERBB4 expression, transgenic mice were bred continuously for a year after which mammary glands were isolated and analyzed. RESULTS: Overexpressing FL CYT-1 isoform resulted in suppression of mammary ductal morphogenesis which was accompanied by decreased number of mammary terminal end buds (TEBs) and Ki-67 positive cells within TEBs, while FL CYT-2 isoform had no effect on ductal growth in pubescent mice. The suppressive ductal phenotype in CYT-1 mice disappeared after mid-pregnancy, and subsequent developmental stages showed no abnormality in mammary gland morphology or function in CYT-1 or CYT-2 transgenic mice. However, sustained expression of FL CYT-1 isoform resulted in formation of neoplastic mammary lesions, suggesting a potential oncogenic function for this isoform. CONCLUSIONS: Together, we present isoform-specific roles of ERBB4 during puberty and early pregnancy, and reveal a novel oncogenic property of CYT-1 ERBB4. The results may be exploited to develop better therapeutic strategies in breast cancer.

Statistical measures of transcriptional diversity capture genomic heterogeneity of cancer.

BACKGROUND: Molecular heterogeneity of tumors suggests the presence of multiple different subclones that may limit response to targeted therapies and contribute to acquisition of drug resistance, but its quantification has remained challenging. RESULTS: We performed simulations to evaluate statistical measures that best capture the molecular diversity within a group of tumors for either continuous (gene expression) or discrete (mutations, copy number alterations) molecular data. Dispersion based metrics in the principal component space best captured the underlying heterogeneity. To demonstrate utility of these measures, we characterized the diversity in transcriptional and genomic profiles of different breast tumor subtypes, and showed that basal-like or triple-negative breast cancers (TNBC) are significantly more heterogeneous molecularly than other subtypes. Our analysis also suggests that transcriptional diversity is a global characteristic of the tumors observed across the majority of molecular pathways. Among basal-like tumors, those that were resistant to multi-agent chemotherapy showed greater transcriptional diversity compared to chemotherapy-sensitive tumors, suggesting that potentially multiple mechanisms may be contributing to chemotherapy resistance. CONCLUSIONS: We proposed and validated measures of transcriptional and genomic diversity that can quantify the molecular diversity of tumors. We applied the new measures to genomic data from breast tumors and demonstrated that basal-like breast cancers are significantly more diverse than other breast cancers. The observation that chemo-resistant tumors are significantly more diverse molecularly than chemosensitive tumors implies that multiple resistance mechanisms may be active, thus limiting the sensitivity and accuracy of predictive markers of chemotherapy response.

Convergent and divergent cellular responses by ErbB4 isoforms in mammary epithelial cells.

UNLABELLED: Associations of ErbB4 (ERBB4/HER4), the fourth member of the EGFR family, with cancer are variable, possibly as a result of structural diversity of this receptor. There are multiple structural isoforms of ERBB4 arising by alternative mRNA splicing, and a subset undergo proteolysis that releases membrane-anchored and soluble isoforms that associate with transcription factors and coregulators to modulate transcription. To compare the differential and common signaling activities of full-length (FL) and soluble intracellular isoforms of ERBB4, four JM-a isoforms (FL and soluble intracellular domain (ICD) CYT-1 and CYT-2) were expressed in isogenic MCF10A cells and their biologic activities were analyzed. Both FL and ICD CYT-2 promoted cell proliferation and invasion, and CYT-1 suppressed cell growth. Transcriptional profiling revealed several new and underexplored ERBB4-regulated transcripts, including: proteases/protease inhibitors (MMP3 and SERPINE2), the YAP/Hippo pathway (CTGF, CYR61, and SPARC), the mevalonate/cholesterol pathway (HMGCR, HMGCS1, LDLR, and DHCR7), and cytokines (IL8, CCL20, and CXCL1). Many of these transcripts were subsequently validated in a luminal breast cancer cell line that normally expresses ERBB4. Furthermore, ChIP-seq experiments identified ADAP1, APOE, SPARC, STMN1, and MXD1 as novel molecular targets of ERBB4. These findings clarify the diverse biologic activities of ERBB4 isoforms, and reveal new and divergent functions. IMPLICATIONS: ErbB4 as a regulator of Hippo and mevalonate pathways provides new insight into milk production and anabolic processes in normal mammary epithelia and cancer.

Global gene expression changes induced by prolonged cold ischemic stress and preservation method of breast cancer tissue.

BACKGROUND: Tissue handling can alter global gene expression potentially affecting the analytical performance of genomic signatures, but such effects have not been systematically evaluated. METHODS: Tissue samples from 11 previously untreated breast tumors were minced and aliquots were either snap frozen or placed in RNAlater immediately or after 20, 40, 60, 120 or 180 min at room temperature. RNA was profiled on Affymetrix HG-U133A arrays. We used probe-set-wise hierarchical models to evaluate the effect of preservation method on transcript expression and linear mixed effects models to assess the effect of cold ischemic delay on the expression of individual probe sets. Gene set enrichment analysis identified pathways overrepresented in the affected transcripts. We combined the levels of 41 most sensitive transcripts to develop an index of ischemic stress. RESULTS: Concordance in global gene expression between the baseline and 40 min delay was higher for samples preserved in RNAlater (average concordance correlation coefficient CCC = 0.92 compared to 0.88 for snap frozen). Overall, 481 transcripts (3%) were significantly affected by the preservation method, most of them involved in processes important in cancer. Prolonged cold ischemic delay of up to 3 h induced marginal global gene expression changes (average CCC = 0.90 between baseline and 3 h delay). However 41 transcripts were significantly affected by cold ischemic delay. Among the induced transcripts were stress response genes, apoptotic response genes; among the downregulated were genes involved in metabolism, protein processing and cell cycle regulation. An index combining the expression levels of these genes was proportional to the cold ischemic delay. CONCLUSIONS: Prolonged cold ischemia induces significant transcriptional changes in a small subset of transcripts in the tissue. Furthermore, the expression level of about 3% of the transcripts is affected by the preservation method. These sensitive transcripts should not be included in genomic signatures for more reliable analytical performance.

Tocotrienol combination therapy results in synergistic anticancer response.

Vitamin E represents a family of compounds that is divided into two subgroups called tocopherols and tocotrienols, which act as important antioxidants that regulate peroxidation reactions and control free-radical production within the body. However, many of the biological effects of vitamin E are mediated independently of its antioxidant activity. Although tocopherols and tocotrienols have the same basic chemical structure characterized by a long phytyl chain attached to a chromane ring, only tocotrienols display potent anticancer activity, by modulating multiple intracellular signaling pathways associated with tumor cell proliferation and survival, and combination therapy with other chemotherapeutic agents result in a synergistic anticancer response. Combination therapy is most effective when tocotrienols are combined with agents that have complementary anticancer mechanisms of action. These findings strongly suggest that the synergistic antiproliferative and apoptotic effects demonstrated by combined low dose treatment of γ-tocotrienol with other chemotherapeutic agents may provide significant health benefits in the prevention and/or treatment of breast cancer in women, while at the same time avoiding tumor resistance and toxic side effects associated with high dose monotherapy.

Synthesis of fluorescent analogues of the anticancer natural products 4-hydroxyphenylmethylene hydantoin and delta-tocotrienol.

4-Hydroxyphenylmethylene hydantoin (PMH, 1), isolated from the Red Sea sponge Hemimycale arabica, and delta-tocotrienol (3), isolated from the tocotrienol-rich fraction of palm oil, are important antimetastatic and antiproliferative natural products that proved effective against metastatic prostate and breast cancers, respectively. New fluorescent derivatives of PMH (2) and delta-tocotrienol (4) were synthesized by Steglich esterification. Both 2 and 4 retained good anti-migratory and antiproliferative activities, respectively. Fluorescent analogues 2 and 4 can be used for the identification of molecular targets of 1 and 3 in tumor cell cultures.