Addressing Barriers and Facilitators to African Americans' and Hispanics' Participation in Clinical and Genomic Research Through a Bioethical Sensitive Video.

Research advances on effective methods to prevent, diagnose, and treat cancer continue to emerge through clinical and genomic research. Most clinical trial and genomic research participants identify as White which limits the generalizability of research findings to non-White populations. With the development and access to technology, digital delivery of salient and tailored health education may provide innovative pathways to increase representation of African Americans (AA) and Hispanics in research. This project focused on the creation of a bioethical sensitive education video aimed at increasing participation in clinical trials and genomic research by bringing together experts from the community, healthcare, biomedical research, and public health. The goal was to utilize existing educational resources to create a tailored message to address AA/Hispanics' beliefs, values, and bioethical concerns related to participation in clinical and genomic research. Models of behavior change and communication theories were leveraged to frame key components of the message, which then informed the framework for the animated video. Development of the video consisted of six iterative phases: 1) writing sessions; 2) storyboarding; 3) animating; 4) screening/revisions; 5) acceptability testing; 6) finalization. The final animated video is approximately 5 min in length and covers several topics including the goal of clinical research, disparities in research participation, bioethical concerns, and genomic research regulations. Increasing AA and Hispanic participation in clinical and genomic research is imperative to achieving health equity. Tailored messages via short videos may assist in addressing the barriers and facilitators towards research participation and increase intentions to enroll in trials.

Whole-exome sequencing of Nigerian benign prostatic hyperplasia reveals increased alterations in apoptotic pathways.

BACKGROUND: Through whole-exome sequencing of 60 formalin-fixed paraffin-embedded Nigerian (NGRn) benign prostatic hyperplasia (BPH) samples, we identified germline and somatic alterations in apoptotic pathways impacting BPH development and progression. Prostate enlargement is a common occurrence in male aging; however, this enlargement can lead to lower urinary tract symptoms that negatively impact quality of life. This impact is disproportionately present in men of African ancestry. BPH pathophysiology is poorly understood and studies examining non-European populations are lacking. METHODS: In this study, NGRn BPH, normal prostate, and prostate cancer (PCa) tumor samples were sequenced and compared to characterize genetic alterations in NGRn BPH. RESULTS: Two hundred and two nonbenign, ClinVar-annotated germline variants were present in NGRn BPH samples. Six genes [BRCA1 (92%), HSD3B1 (85%), TP53 (37%), PMS2 (23%), BARD1 (20%), and BRCA2 (17%)] were altered in at least 10% of samples; however, compared to NGRn normal and tumor, the frequency of alterations in BPH samples showed no significant differences at the gene or variant level. BRCA2_rs11571831 and TP53_rs1042522 germline alterations had a statistically significant co-occurrence interaction in BPH samples. In at least two BPH samples, 173 genes harbored somatic variants known to be clinically actionable. Three genes (COL18A1, KIF16B, and LRP1) showed a statistically significant (p < 0.05) higher frequency in BPH. NGRn BPH also had five gene pairs (PKD1/KIAA0100, PKHD1/PKD1, DNAH9/LRP1B, NWD1/DCHS2, and TCERG1/LMTK2) with statistically significant co-occurring interactions. Two hundred and seventy-nine genes contained novel somatic variants in NGRn BPH. Three genes (CABP1, FKBP1C, and RP11-595B24.2) had a statistically significant (p < 0.05) higher alteration frequency in NGRn BPH and three were significantly higher in NGRn tumor (CACNA1A, DMKN, and CACNA2D2). Pairwise Fisher's exact tests showed 14 gene pairs with statistically significant (p < 0.05) interactions and four interactions approaching significance (p < 0.10). Mutational patterns in NGRn BPH were similar to COSMIC (Catalog of Somatic Mutations in Cancer) signatures associated with aging and dysfunctional DNA damage repair. CONCLUSIONS: NGRn BPH contained significant germline alteration interactions (BRCA2_rs11571831 and TP53_rs1042522) and increased somatic alteration frequencies (LMTK2, LRP1, COL18A1, CABP1, and FKBP1C) that impact apoptosis. Normal prostate development is maintained by balancing apoptotic and proliferative activity. Dysfunction in either mechanism can lead to abnormal prostate growth. This work is the first to examine genomic sequencing in NGRn BPH and provides data that fill known gaps in the understanding BPH and how it impacts men of African ancestry.

Immune Profile of Exosomes in African American Breast Cancer Patients Is Mediated by Kaiso/THBS1/CD47 Signaling.

African American (AA) women with breast cancer are more likely to have higher inflammation and a stronger overall immune response, which correlate with poorer outcomes. In this report, we applied the nanostring immune panel to identify differences in inflammatory and immune gene expression by race. We observed a higher expression of multiple cytokines in AA patients compared to EA patients, with high expression of CD47, TGFB1, and NFKB1 associated with the transcriptional repressor Kaiso. To investigate the mechanism associated with this expression pattern, we observed that Kaiso depletion results in decreased expression of CD47, and its ligand SIRPA. Furthermore, Kaiso appears to directly bind to the methylated sequences of the THBS1 promotor and repress gene expression. Similarly, Kaiso depletion attenuated tumor formation in athymic nude mice, and these Kaiso-depleted xenograft tissues showed significantly higher phagocytosis and increased infiltration of M1 macrophages. In vitro validation using MCF7 and THP1 macrophages treated with Kaiso-depleted exosomes showed a reduced expression of immune-related markers (CD47 and SIRPA) and macrophage polarization towards the M1 phenotype compared to MCF7 cells treated with exosomes isolated from high-Kaiso cells. Lastly, analysis of TCGA breast cancer patient data demonstrates that this gene signature is most prominent in the basal-like subtype, which is more frequently observed in AA breast cancer patients.

Integrating Pharmacogenomics Data-Driven Computational Drug Prediction with Single-Cell RNAseq to Demonstrate the Efficacy of a NAMPT Inhibitor against Aggressive, Taxane-Resistant, and Stem-like Cells in Lethal Prostate Cancer.

Metastatic prostate cancer/PCa is the second leading cause of cancer deaths in US men. Most early-stage PCa are dependent on overexpression of the androgen receptor (AR) and, therefore, androgen deprivation therapies/ADT-sensitive. However, eventual resistance to standard medical castration (AR-inhibitors) and secondary chemotherapies (taxanes) is nearly universal. Further, the presence of cancer stem-like cells (EMT/epithelial-to-mesenchymal transdifferentiation) and neuroendocrine PCa (NEPC) subtypes significantly contribute to aggressive/lethal/advanced variants of PCa (AVPC). In this study, we introduced a pharmacogenomics data-driven optimization-regularization-based computational prediction algorithm ("secDrugs") to predict novel drugs against lethal PCa. Integrating secDrug with single-cell RNA-sequencing/scRNAseq as a 'Double-Hit' drug screening tool, we demonstrated that single-cells representing drug-resistant and stem-cell-like cells showed high expression of the NAMPT pathway genes, indicating potential efficacy of the secDrug FK866 which targets NAMPT. Next, using several cell-based assays, we showed substantial impact of FK866 on clinically advanced PCa as a single agent and in combination with taxanes or AR-inhibitors. Bulk-RNAseq and scRNAseq revealed that, in addition to NAMPT inhibition, FK866 regulates tumor metastasis, cell migration, invasion, DNA repair machinery, redox homeostasis, autophagy, as well as cancer stemness-related genes, HES1 and CD44. Further, we combined a microfluidic chip-based cell migration assay with a traditional cell migration/'scratch' assay and demonstrated that FK866 reduces cancer cell invasion and motility, indicating abrogation of metastasis. Finally, using PCa patient datasets, we showed that FK866 is potentially capable of reversing the expression of several genes associated with biochemical recurrence, including IFITM3 and LTB4R. Thus, using FK866 as a proof-of-concept candidate for drug repurposing, we introduced a novel, universally applicable preclinical drug development pipeline to circumvent subclonal aggressiveness, drug resistance, and stemness in lethal PCa.

MicroRNAs within the Basal-like signature of Quadruple Negative Breast Cancer impact overall survival in African Americans.

We previously found that QNBC tumors are more frequent in African Americans compared to TNBC tumors. To characterize this subtype further, we sought to determine the miRNA-mRNA profile in QNBC patients based on race. Both miRNA and mRNA expression data were analyzed from TCGA and validated using datasets from the METABRIC, TCGA proteomic, and survival analysis by KMPLOT. miRNA-mRNAs which include FOXA1 and MYC (mir-17/20a targets); GATA3 and CCNG2 (mir-135b targets); CDKN2A, CDK6, and B7-H3 (mir-29c targets); and RUNX3, KLF5, IL1-ß, and CTNNB1 (mir-375 targets) were correlated with basal-like and immune subtypes in QNBC patients and associated with a worse survival. Thus, QNBC tumors have an altered gene signature implicated in racial disparity and poor survival.

Whole-exome Sequencing of Nigerian Prostate Tumors from the Prostate Cancer Transatlantic Consortium (CaPTC) Reveals DNA Repair Genes Associated with African Ancestry.

In this study, we used whole-exome sequencing of a cohort of 45 advanced-stage, treatment-naïve Nigerian (NG) primary prostate cancer tumors and 11 unmatched nontumor tissues to compare genomic mutations with African American (AA) and European American (EA) The Cancer Genome Atlas (TCGA) prostate cancer. NG samples were collected from six sites in central and southwest Nigeria. After whole-exome sequencing, samples were processed using GATK best practices. BRCA1 (100%), BARD1 (45%), BRCA2 (27%), and PMS2(18%) had germline alterations in at least two NG nontumor samples. Across 111 germline variants, the AA cohort reflected a pattern [BRCA1 (68%), BARD1 (34%), BRCA2 (28%), and PMS2 (16%)] similar to NG samples. Of the most frequently mutated genes, BRCA1 showed a statistically (P ≤ 0.05) higher germline mutation frequency in men of African ancestry (MAA) and increasing variant frequency with increased African ancestry. Disaggregating gene-level mutation frequencies by variants revealed both ancestry-linked and NG-specific germline variant patterns. Driven by rs799917 (T>C), BRCA1 showed an increasing mutation frequency as African ancestry increased. BRCA2_rs11571831 was present only in MAA, and BRCA2_rs766173 was elevated in NG men. A total of 133 somatic variants were present in 26 prostate cancer-associated genes within the NG tumor cohort. BRCA2 (27%), APC (20%), ATM (20%), BRCA1 (13%), DNAJC6 (13%), EGFR (13%), MAD1L1 (13%), MLH1 (11%), and PMS2 (11%) showed mutation frequencies >10%. Compared with TCGA cohorts, NG tumors showed statistically significant elevated frequencies of BRCA2, APC, and BRCA1. The NG cohort variant pattern shared similarities (cosign similarities ≥0.734) with Catalogue of Somatic Mutations in Cancer signatures 5 and 6, and mutated genes showed significant (q < 0.001) gene ontology (GO) and functional enrichment in mismatch repair and non-homologous repair deficiency pathways. Here, we showed that mutations in DNA damage response genes were higher in NG prostate cancer samples and that a portion of those mutations correlate with African ancestry. Moreover, we identified variants of unknown significance that may contribute to population-specific routes of tumorigenesis and treatment. These results present the most comprehensive characterization of the NG prostate cancer exome to date and highlight the need to increase diversity of study populations. Significance: MAA have higher rates of prostate cancer incidence and mortality, however, are severely underrepresented in genomic studies. This is the first study utilizing whole-exome sequencing in NG men to identify West African ancestry-linked variant patterns that impact DNA damage repair pathways.

Identification of Distinct Heterogenic Subtypes and Molecular Signatures Associated with African Ancestry in Triple Negative Breast Cancer Using Quantified Genetic Ancestry Models in Admixed Race Populations.

Triple negative breast cancers (TNBCs) are molecularly heterogeneous, and the link between their aggressiveness with African ancestry is not established. We investigated primary TNBCs for gene expression among self-reported race (SRR) groups of African American (AA, n = 42) and European American (EA, n = 33) women. RNA sequencing data were analyzed to measure changes in genome-wide expression, and we utilized logistic regressions to identify ancestry-associated gene expression signatures. Using SNVs identified from our RNA sequencing data, global ancestry was estimated. We identified 156 African ancestry-associated genes and found that, compared to SRR, quantitative genetic analysis was a more robust method to identify racial/ethnic-specific genes that were differentially expressed. A subset of African ancestry-specific genes that were upregulated in TNBCs of our AA patients were validated in TCGA data. In AA patients, there was a higher incidence of basal-like two tumors and altered TP53, NFB1, and AKT pathways. The distinct distribution of TNBC subtypes and altered oncologic pathways show that the ethnic variations in TNBCs are driven by shared genetic ancestry. Thus, to appreciate the molecular diversity of TNBCs, tumors from patients of various ancestral origins should be evaluated.

Quadruple Negative Breast Cancers (QNBC) Demonstrate Subtype Consistency among Primary and Recurrent or Metastatic Breast Cancer.

PURPOSE: Despite the availability of current standards of care treatments for triple negative breast cancer (TNBC), many patients still die from this disease. Quadruple negative tumors, which are TNBC tumors that lack androgen receptor (AR), represent a more aggressive subtype of TNBC; however, the molecular features are not well understood. METHODS: Immunohistochemistry of estrogen receptor (ER), progesterone receptor (PR), HER2, and AR was determined in 244 primary and 630 recurrent/metastatic site biopsies. Expression was correlated with a panel of 25 cancer-related genes and proteins by IHC and in situ hybridization (ISH). RESULTS: We observed that 80.2% (65 of 81) of primary TNBC tumors and 75.7% (159 of 210) of recurrent/metastatic TNBC tumors are QNBC. Bivariate fit analysis demonstrated that QNBC (n = 224) significantly (P < .03) correlated with younger aged patients at initial biopsy compared to AR positive TNBC patients (n = 51). In paired primary tissue samples and primary to recurrent/metastatic samples, at least 70% Luminal, HER2 enriched, and QNBC subtype did not change molecular profile. But, TNBC seems to be the "unstable" subtype. Within the total cohort, discordance in molecular profiles was identified in both synchronous (20%) and asynchronous (21%) intra-individual analyses. Irrespective of sample type, (Synchronous or Asynchronous), QNBC demonstrated higher concordant than TNBC. IHC and ISH results of the cancer related genes, demonstrated that gene/protein expression differ by molecular profile: TNBC (HR-/HER2-, AR+) and QNBC (HR-/HER2-, AR-). IHC in metastatic tumors, showed that the percentage of tumors positive of EGFR were higher, while PTEN and TLE3 were lower in QNBC compared to TNBC. CONCLUSION: Standard treatment of Breast Cancer (BC) relies on reliable assessment by IHC analysis of ER, PR, and HER2. Our analyses suggest that the heterogeneity of TNBC is at least partially associated with the presence or absence of AR expression, suggesting that QNBC should be considered as a clinically relevant BC subtype. IHC analysis of AR appears to be a practical assay to determine the most aggressive TNBC subtypes and identifies tumors that could benefit from available targeted therapies.

AR negative triple negative or "quadruple negative" breast cancers in African American women have an enriched basal and immune signature.

There is increasing evidence that Androgen Receptor (AR) expression has prognostic usefulness in Triple negative breast cancer (TNBC), where tumors that lack AR expression are considered "Quadruple negative" Breast Cancers ("QNBC"). However, a comprehensive analysis of AR expression within all breast cancer subtypes or stratified by race has not been reported. We assessed AR mRNA expression in 925 tumors from The Cancer Genome Atlas (TCGA), and 136 tumors in 2 confirmation sets. AR protein expression was determined by immunohistochemistry in 197 tumors from a multi-institutional cohort, for a total of 1258 patients analyzed. Cox hazard ratios were used to determine correlations to PAM50 breast cancer subtypes, and TNBC subtypes. Overall, AR-negative patients are diagnosed at a younger age compared to AR-positive patients, with the average age of AA AR-negative patients being, 49. AA breast tumors express AR at lower rates compared to Whites, independent of ER and PR expression (p<0.0001). AR-negative patients have a (66.60; 95% CI, 32-146) odds ratio of being basal-like compared to other PAM50 subtypes, and this is associated with an increased time to progression and decreased overall survival. AA "QNBC" patients predominately demonstrated BL1, BL2 and IM subtypes, with differential expression of E2F1, NFKBIL2, CCL2, TGFB3, CEBPB, PDK1, IL12RB2, IL2RA, and SOS1 genes compared to white patients. Immune checkpoint inhibitors PD-1, PD-L1, and CTLA-4 were significantly upregulated in both overall "QNBC" and AA "QNBC" patients as well. Thus, AR could be used as a prognostic marker for breast cancer, particularly in AA "QNBC" patients.

African Americans with pancreatic ductal adenocarcinoma exhibit gender differences in Kaiso expression.

Kaiso, a bi-modal transcription factor, regulates gene expression, and is elevated in breast, prostate, and colon cancers. Depletion of Kaiso in other cancer types leads to a reduction in markers for the epithelial-mesenchymal transition (EMT) (Jones et al., 2014), however its clinical implications in pancreatic ductal adenocarcinoma (PDCA) have not been widely explored. PDCA is rarely detected at an early stage but is characterized by rapid progression and invasiveness. We now report the significance of the subcellular localization of Kaiso in PDCAs from African Americans. Kaiso expression is higher in the cytoplasm of invasive and metastatic pancreatic cancers. In males, cytoplasmic expression of Kaiso correlates with cancer grade and lymph node positivity. In male and female patients, cytoplasmic Kaiso expression correlates with invasiveness. Also, nuclear expression of Kaiso increases with increased invasiveness and lymph node positivity. Further, analysis of the largest PDCA dataset available on ONCOMINE shows that as Kaiso increases, there is an overall increase in Zeb1, which is the inverse for E-cadherin. Hence, these findings suggest a role for Kaiso in the progression of PDCAs, involving the EMT markers, E-cadherin and Zeb1.

Kaiso, a transcriptional repressor, promotes cell migration and invasion of prostate cancer cells through regulation of miR-31 expression.

Kaiso, a member of the BTB/POZ zinc finger protein family, functions as a transcriptional repressor by binding to sequence-specific Kaiso binding sites or to methyl-CpG dinucleotides. Previously, we demonstrated that Kaiso overexpression and nuclear localization correlated with the progression of prostate cancer (PCa). Therefore, our objective was to explore the molecular mechanisms underlying Kaiso-mediated PCa progression. Comparative analysis of miRNA arrays revealed that 13 miRNAs were significantly altered (> 1.5 fold, p < 0.05) in sh-Kaiso PC-3 compared to sh-Scr control cells. Real-time PCR validated that three miRNAs (9, 31, 636) were increased in sh-Kaiso cells similar to cells treated with 5-aza-2'-deoxycytidine. miR-31 expression negatively correlated with Kaiso expression and with methylation of the miR-31 promoter in a panel of PCa cell lines. ChIP assays revealed that Kaiso binds directly to the miR-31 promoter in a methylation-dependent manner. Over-expression of miR-31 decreased cell proliferation, migration and invasiveness of PC-3 cells, whereas cells transfected with anti-miR-31 restored proliferation, migration and invasiveness of sh-Kaiso PC-3 cells. In PCa patients, Kaiso high/miR-31 low expression correlated with worse overall survival relative to each marker individually. In conclusion, these results demonstrate that Kaiso promotes cell migration and invasiveness through regulation of miR-31 expression.

Inhibition of the Warburg effect with a natural compound reveals a novel measurement for determining the metastatic potential of breast cancers.

Metabolism is an important differentiating feature of cancer cells. Lactate dehydrogenases (LDH) A/B are metabolically important proteins and are involved in the critical step of inter-conversion of lactate to pyruvate. Panepoxydone (PP), a natural NF-kB inhibitor, significantly reduces the oxygen consumption and lactate production of MCF-7 and triple negative (MDA-MB-231, MDA-MB-468 and MDA-MB-453) breast cancer cells. We further observed that PP inhibited mitochondrial membrane potential and the ATP synthesis using flow cytometry. PP also up-regulated LDH-B and down-regulated LDH-A expression levels in all breast cancer cells to similar levels observed in HMEC cells. Over-expression of LDH-B in cancer cell lines leads to enhanced apoptosis, mitochondrial damage, and reduced cell migration. Analyzing the patient data set GDS4069 available on the GEO website, we observed 100% of non TNBC and 60% of TNBC patients had less LDH-B expression than LDH-A expression levels. Herein we report a new term called Glycolytic index, a novel method to calculate utilization of oxidative phosphorylation in breast cancer cells through measuring the ratio of the LDH-B to LDH-A. Furthermore, inhibitors of NF-kB could serve as a therapeutic agent for targeting metabolism and for the treatment of triple negative breast cancer.

MicroRNA profiling of novel African American and Caucasian Prostate Cancer cell lines reveals a reciprocal regulatory relationship of miR-152 and DNA methyltranferase 1.

miRNA expression in African American compared to Caucasian PCa patients has not been widely explored. Herein, we probed the miRNA expression profile of novel AA and CA derived prostate cancer cell lines. We found a unique miRNA signature associated with AA cell lines, independent of tumor status. Evaluation of the most differentially expressed miRNAs showed that miR-132, miR-367b, miR-410, and miR-152 were decreased in more aggressive cells, and this was reversed after treatment of the cells with 5-aza-2'-deoxycytidine. Sequencing of the miR-152 promoter confirmed that it was highly methylated. Ectopic expression of miR-152 resulted in decreased growth, migration, and invasion. Informatics analysis of a large patient cohort showed that decreased miR-152 expression correlated with increased metastasis and a decrease in biochemical recurrence free survival. Analysis of 39 prostate cancer tissues with matched controls (20 AA and 19 CA), showed that 50% of AA patients had statistically significant lower miR-152 expression compared to only 35% of CA patients. Ectopic expression of miR-152 in LNCaP, PC-3, and MDA-PCa-2b cells down-regulated DNA (cytosine-5)-methyltransferase 1 (DNMT1) through direct binding in the DNMT1 3'UTR. There appeared to be a reciprocal regulatory relationship of miR-152/DNMT1 expression, as cells treated with siRNA DNMT1 caused miR-152 to be re-expressed in all cell lines. In summary, these results demonstrate that epigenetic regulation of miR-152/DNMT1 may play an important role in multiple events that contribute to the aggressiveness of PCa tumors, with an emphasis on AA PCa patients .

Panepoxydone targets NF-kB and FOXM1 to inhibit proliferation, induce apoptosis and reverse epithelial to mesenchymal transition in breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is a highly diverse group that is associated with an aggressive phenotype. Its treatment has been challenging due to its heterogeneity and absence of well-defined molecular targets. Thus, there is an urgent need to identify novel agents with therapeutic application. NF-κB is over-expressed in many breast cancers; thus, inactivation of the NF-κB pathway could serve as a therapeutic target. Here we report for the first time the anti-tumor activity of panepoxydone (PP), a NF-κB inhibitor isolated from an edible mushroom, in several breast cancer cell lines. METHODS: We investigated the effects of PP on cell growth, migration-invasion, apoptosis and EMT-related proteins expression in MCF-7 and TNBC cell lines MDA-MB-231, MDA-MB-468 and MDA-MB-453. RESULTS: Significant antitumor activity was seen in all cell lines, with differential responses noted in cell-line specific manner. Treatment with PP resulted in significant cytotoxicity, decreased invasion, migration and increased apoptosis in all cell lines tested. Up-regulation of Bax and cleaved PARP and down-regulation of Bcl-2, survivin, cyclin D1 and caspase 3 were noted in PP-treated breast cancer cells. The antitumor effect of PP appeared related to its ability to inhibit the phosphorylation of inhibitor of NF-κB (IκBα) with cytoplasmic accumulation. PP treatment also down-regulated FOXM1 which resulted in a reversal of EMT. Similar results were obtained after silencing of NF-kB and FOXM1. CONCLUSION: Altogether, these studies show, for the first time the antitumor activity of PP against breast cancer cells, in particular TNBC cells. Furthermore, it highlights the concept that optimal treatment of TNBC warrants attention to the differential sensitivity of various TNBC subtypes to therapeutic agents. These results suggest that the PP may be a potentially effective chemopreventive or therapeutic agent against breast cancer. However, additional studies are required to more fully elucidate the mechanism of antitumor effect of PP.

Nuclear localization of Kaiso promotes the poorly differentiated phenotype and EMT in infiltrating ductal carcinomas.

The expression and biological consequences of Kaiso, a novel bi-modal transcription factor, in infiltrating ductal carcinomas (IDCs) have not been widely investigated. In the present study, we determined Kaiso expression and subcellular localization in 146 normal tissues, 376 IDCs, and 85 lymph node metastases. In IDCs, there was higher Kaiso expression in both the cytoplasmic and nuclear compartments, which correlated with age <48 (cytoplasmic p < 0.0093; nuclear p < 0.0001) and moderate differentiation (cytoplasmic p < 0.0042; nuclear p < 0.0001), as determined by Chi square analysis. However, only nuclear Kaiso correlated with poor prognostic factors, i.e., race (African Americans) (p < 0.0001), poor differentiation (p < 0.0001), and metastases (p < 0.0001). Nuclear Kaiso was also associated with worse overall survival (p < 0.0019), with African American patients displaying worse survival rates relative to Caucasian patients (p < 0.029). MCF-7 (non-metastatic), MDA-MB-468 (few metastases), and MDA-MB-231 (highly metastatic) breast cancer cells demonstrated increasing Kaiso levels, with more nuclear localization in the highly metastatic cell line. Over-expression of Kaiso in MCF-7 cells increased cell migration and invasion, but treatment of MDA-MB-468 and MDA-MB-231 cells with si-Kaiso decreased cell migration and invasion and induced expression of E-cadherin RNA and protein. E-cadherin re-expression was associated with a reversal of mesenchymal associated cadherins, N-cadherin and cadherin 11, as well as decreased vitamin expression. Further, Kaiso directly bound to methylated sequences in the E-cadherin promoter, an effect prevented by 5-aza-2-deoxycytidine. Immunofluorescence co-staining of poorly differentiated IDCs demonstrated that nuclear Kaiso is associated with a loss of E-cadherin expression. These findings support a role for Kaiso in promoting aggressive breast tumors.

Elevated serum P1NP predicts development of bone metastasis and survival in early-stage breast cancer.

Bone is the most common site of metastasis of breast cancer, affecting most women with advanced disease. Procollagen type I N-terminal propeptide (P1NP), osteocalcin, CTX, and IL-6 are markers of bone turnover. Our objective was to determine whether serum levels of these proteins have clinical utility as predictors of breast cancer metastasis to bone. Blood was collected before treatment from 164 patients with stage I-III breast cancer from September 2001 to December 2008. Serum levels of P1NP, CTX, IL-6, and OC were measured using an automated immunoassay system. Correlations of the levels of these markers with time to bone metastasis development and with overall survival (OS) rate were assessed using Cox proportional hazards regression analysis and the Kaplan-Meier method. Fifty-five patients with stage I-III disease at the time of blood sample collection subsequently experienced metastasis to bone. A baseline P1NP level of at least 75 ng/mL predicted increased risk of bone metastasis (hazard ratio, 2.7 [95 % confidence interval, 1.2-6.0]; P = 0.031) and a poor OS rate (P = 0.031). Serum P1NP levels at or above 75 ng/mL correlate with a short time to development of bone metastasis and low overall survival in patients with stage I-III breast cancer.

Receptor tyrosine kinase ErbB2 translocates into mitochondria and regulates cellular metabolism.

It is well known that ErbB2, a receptor tyrosine kinase, localizes to the plasma membrane. Here we describe a novel observation that ErbB2 also localizes in mitochondria of cancer cells and patient samples. We found that ErbB2 translocates into mitochondria through association with mtHSP70. Additionally, mitochondrial ErbB2 (mtErbB2) negatively regulates mitochondrial respiratory functions. Oxygen consumption and activities of complexes of the mitochondrial electron transport chain were decreased in mtErbB2-overexpressing cells. Mitochondrial membrane potential and cellular ATP levels were also decreased. In contrast, mtErbB2 enhanced cellular glycolysis. The translocation of ErbB2 and its impact on mitochondrial function are kinase dependent. Interestingly, cancer cells with higher levels of mtErbB2 were more resistant to the ErbB2-targeting antibody trastuzumab. Our study provides a novel perspective on the metabolic regulatory function of ErbB2 and reveals that mtErbB2 has an important role in the regulation of cellular metabolism and cancer cell resistance to therapeutics.

Her2-positive breast cancer: herceptin and beyond.

Breast cancer accounts for approximately 30% of all new cancer cases each year, with an annual incidence of approximately 200,000. Additionally, almost 25% of breast cancers are noted to overexpress Her2, which is an epidermal growth factor receptor. Overexpression of Her2 has been associated with a more aggressive phenotype with decreased survival. Trastuzumab, a recombinant monoclonal antibody against the Her2 receptor, is the only FDA-approved targeted agent for treatment of Her2-overexpressing breast cancer. However, despite the great success achieved with trastuzumab, many women will either not respond or eventually progress despite trastuzumab treatment. As a result, significant efforts have been applied to finding other therapies besides trastuzumab for the treatment of Her2-positive breast cancer. Work has been directed at trying to elucidate the exact mechanism of resistance to trastuzumab and identifying ways to overcome them, at increasing the efficacy of trastuzumab by combining it with other therapeutic agents and at investigating other novel agents.

Emerging agents in the treatment of anthracycline- and taxane-refractory metastatic breast cancer.

Despite the recent trend toward treatment of early stage breast cancer aggressively with anthracyclines and taxanes, nearly half of those women will have metastatic recurrence. Moreover, because of the increasing prior exposure to these drugs, far more women facing first-line therapy for recurrent disease will now have developed anthracycline- and taxane-refractory metastatic breast cancer (ATRMBC), presenting a major therapeutic challenge. A number of established drugs are showing promise in this setting: capecitabine alone or combined with lapatinib; gemcitabine; vinorelbine; and oxaliplatin. At the same time, a variety of new drugs are emerging for potential use in ATRMBC. Among the drugs in clinical development that have shown promising activity include novel classes of compounds (camptothecins and epothilones), newer members of established classes (pemetrexed and vinflunine), and agents with novel mechanisms of action (the mitosis inhibitor E7389 and the ascidian-derived anticancer compound trabectedin). Several molecularly targeted agents are also being evaluated in ATRMBC, including interleukin-2 receptor-binding denileukin diftitox, and 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), which inhibits the protein chaperone heat shock protein 90.