Citations

Abstract Background: NSABP P1 and P2 are the largest prevention trials of tamoxifen and raloxifene in women at high risk of developing breast cancer. Both agents have been shown to decrease, but not eliminate breast cancer risk in these women. The goal of our study was to identify single nucleotide polymorphisms (SNPs) associated with development of breast cancer in high-risk women treated with tamoxifen or raloxifene. Methods: We performed a nested matched case-control genome-wide association study (GWAS) in subjects entered on the P2, which included only postmenopausal women, and subjects at least 50 years old entered on the P1 phase III prevention trials who were treated with tamoxifen or raloxifene. Cases were defined as subjects who developed invasive breast cancer or ductal carcinoma in situ and each was matched to two controls based on trial (P1, P2), age at trial entry, Gail risk score, history of lobular carcinoma in situ, and time on study. The study was restricted to the 94% of subjects self-identified as Caucasian. Genotyping was performed with the Illumina Human610-Quad chip. Eigenstrat analyses were performed to control for population stratification. SNPs were imputed within 200 Kb on either side of regions containing SNPs with p ≤ 3E-05. CYP2D6 genotyping was performed on all subjects. Results: The GWAS included 592 cases and 1171 controls. 547,356 SNPs were used in the analyses after removing 14,019 for call rates less than 98%, 30,560 for minor allele frequencies <1%, and 301 with Hardy-Weinberg equilibrium p-value <1E-06. Eigenvectors did not alter the results when used as covariates. Eleven genotyped SNPs with p-values ranging from 2.12E-06 to 9.98E-06 (conditional logistic regression with eigenvector adjustment) were identified; and 22 SNPs with p < 4E-05 were identified by imputation around these SNPs on chromosomes (Chr) 4, 8, 9, 13, 16 and 22, which were then genotyped with the Invader platform. Following imputation and genotyping, initial functional genomic studies have focused on 8 SNPs on Chr 16 (p-values 1.81E-06 to 9.55E-06, with the lowest p-value for an imputed and fine mapped SNP), all of which were in ZNF423, a gene known to encode a protein that associates with SMAD family members. In lymphoblastoid cell lines genotyped for the 2 top SNPs in ZNF423 (rs8060157 and rs11076499) and stably transfected with estrogen receptor-α, the variant and wild type SNPs displayed striking differences in estradiol-induced expression of ZNF423, SMAD3, BRCA1, and BRCA2. Conclusions: This GWAS revealed SNPs associated with the development of breast cancer in high-risk women treated with tamoxifen or raloxifene. Genome-wide significance was not achieved, but SNPs in ZNF423 identified on Chr 16 were associated with differential expression of ZNF423, SMAD3, BRCA1, and BRCA2 in response to estradiol in vitro, providing direction for future research. (Supported in part by NIH grants U01GM61388, U01GM63173, P50CA116201, U10CA77202, U10CA37377, U10CA69974, U24CA114732, and the Biobank Japan Project funded by the Ministry of Education, Culture, Sports, Science and Technology, Japan) Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD05-02.

Abstract The mitochondrial transcription factor A (TFAM) is required for mitochondrial DNA (mtDNA) replication and transcription. Disruption of TFAM results in heart failure and premature aging in mice. But very little is known about the role of TFAM in cancer development. Here, we report the identification of frequent frameshift mutations in the coding mononucleotide repeat of TFAM in sporadic colorectal cancer (CRC) cell lines and in primary tumors with microsatellite instability (MSI), but not in microsatellite stable (MSS) CRC cell lines and tumors. The presence of the TFAM truncating mutation, in CRC cells with MSI, reduced the TFAM protein level in vivo and in vitro and correlated with mtDNA depletion. Furthermore, forced overexpression of wild-type TFAM in RKO cells carrying a TFAM truncating mutation suppressed cell proliferation and inhibited RKO cell-induced xenograft tumor growth. Moreover, these cells showed more susceptibility to cisplatin-induced apoptosis due to an increase of cytochrome b (Cyt b) expression and its release from mitochondria. An interaction assay between TFAM and the heavy-strand promoter (HSP) of mitochondria revealed that mutant TFAM exhibited reduced binding to HSP, leading to reduction in Cyt b transcription. Collectively, these data provide evidence that a high incidence of TFAM truncating mutations leads to mitochondrial copy number reduction and mitochondrial instability, distinguishing most CRC with MSI from MSS CRC. These mutations may play an important role in tumorigenesis and cisplatin-induced apoptotic resistance of most microsatellite-unstable CRCs. Cancer Res; 71(8); 2978–87. ©2011 AACR.

Nitric oxide (NO) regulates the function of perivascular cells (pericytes), including hepatic stellate cells (HSC), mainly by activating cGMP and cGMP-dependent kinase (PKG) via NO/cGMP paracrine signaling. Although PKG is implicated in integrin-mediated cell adhesion to extracellular matrix, whether or how PKG signaling regulates the assembly of focal adhesion complexes (FA) and migration of HSC is not known. With the help of complementary molecular and cell biological approaches, we demonstrate here that activation of PKG signaling in HSC inhibits vascular tubulogenesis, migration/chemotaxis, and assembly of mature FA plaques, as assessed by vascular tubulogenesis assays and immunofluorescence localization of FA markers such as vinculin and vasodilator-stimulated phosphoprotein (VASP). To determine whether PKG inhibits FA assembly by phosphorylation of VASP at Ser-157, Ser-239, and Thr-278, we mutated these putative phosphorylation sites to alanine (VASP3A, phosphoresistant mutant) or aspartic acid (VASP3D, phosphomimetic), respectively. Data generated from these two mutants suggest that the effect of PKG on FA is independent of these three phosphorylation sites. In contrast, activation of PKG inhibits the activity of small GTPase Rac1 and its association with the effector protein IQGAP1. Moreover, PKG activation inhibits the formation of a trimeric protein complex containing Rac1, IQGAP1, and VASP. Finally, we found that expression of a constitutively active Rac1 mutant abolishes the inhibitory effects of PKG on FA formation. In summary, our data suggest that activation of PKG signaling in pericytes inhibits FA formation by inhibiting Rac1.

AbstractCCN1, also known as CYR61, is a survival and proangiogenic factor overexpressed in about 30% of invasive breast carcinomas, and particularly in triple-negative breast carcinomas (TNBC). CCN1 expression in breast cancer promotes tumorigenicity, metastasis, antihormone, and chemoresistance. TNBCs often develop bone metastasis, thus the vast majority of patients receive bisphosphonate treatment as a companion to chemotherapy. Zoledronic acid (ZOL), a bisphosphonate currently in use, inhibits bone resorption, prevents development of new osteolytic lesions induced by tumor metastasis, and has a direct antitumor activity in breast cancer cells and tumors. We have shown that ZOL inhibits anchorage independent growth as well as branching and morphogenesis in CCN1 overexpressing cells. However, the mechanism is not yet well understood. In this study, we investigate the effect of ZOL in breast cancer cells with high and undetectable CCN1 expression levels. We show that CCN1-expressing cells are more sensitive to ZOL, that ZOL induces downregulation of the CCN1 promoter activity and CCN1 protein expression in a dose-dependent manner, and that ZOL is associated with a decrease in phosphorylated Akt and translocation of FOXO3a, a negative regulator of CCN1 expression, to the nucleus. Deletion of the FOXO3a binding site in the CCN1 promoter prevents ZOL inhibition of the CCN1 promoter activity showing that FOXO3a transcriptional activation is necessary for ZOL to induce CCN1 inhibition. This study provides evidence that ZOL targets the proangiogenic factor (CCN1) through FOXO3a and reveals a new mechanism of ZOL action in breast cancer cells. Mol Cancer Ther; 10(5); 732–41. ©2011 AACR.

Abstract Purpose: This study uses spatially accurate image-guided tissue analysis to evaluate the correlation between Perfusion MRI (pMRI) and tissue microvascular density parameters as markers for tumor recurrence and treatment response in high-grade glioma (HGG). Methods: Following Institutional Review Board approval, we recruited previously treated (including chemo-radiation therapy) HGG patients undergoing surgical re-resection of new enhancing lesions on surveillance MRI. Preoperatively, we acquired pMRI and contrast-enhanced stereotactic T1-Weighted (T1W) MRI data sets. Intraoperatively, we recorded stereotactic locations of multiple biopsies based on T1W data sets that guided surgery. Following surgery, we coregistered pMRI and T1W data sets to calculate localized relative cerebral blood volume (rCBV) for each stereotactic specimen location. For each specimen, we performed 1) standard H&E staining to diagnose tumor recurrence or post-treatment radiation effect (PTRE); and 2) immunohistochemical analysis with CD34 to highlight tissue vessels. We analyzed CD-34 stained slides with Axiovision Automeasure 3.4 (Zeiss, Germany) to calculate both total microvessel number (MVN) and total microvessel area (MVA), each normalized to total slide specimen area (μm2). We calculated Pearson correlations to establish relationships between a) rCBV and MVN; and b) rCBV and MVA. We also performed t-test to compare MVN, MVA, and rCBV values between tumor and PTRE samples. A neuroradiologist performed all coregistration and pMRI calculations, and a neuropathologist analyzed all tissue specimens, without knowledge of corresponding data. A biostatistician performed all statistical comparisons. Results: In this preliminary study, we included 16 tissue specimens (from 8 subjects), each diagnosed as either tumor (n=7) or PTRE (n=9). We successfully calculated localized rCBV and determined both MVA and MVN for each specimen. The rCBV values showed highest correlation with total vessel area (MVA) (r=0.65, p=0.007) and slightly less correlation with vessel number (MVN) (r=0.52, p=0.04). Tumor showed significantly higher values than PTRE for all parameters: rCBV (1.87 + 0.82 versus 0.78 + 0.2, p=.002); MVA (0.22 + 0.03 versus 0.04 + 0.007, p=0.0001); and MVN (0.0068 + 0.001 versus. 0.0016 + 0.0006, p=0.0004). Conclusion: These preliminary results show the promise of Perfusion MRI to non-invasively estimate tissue microvessel density and distinguish tumor recurrence from treatment effects. The current study is ongoing to confirm results in a larger patient population. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3748.

In comparing gene expression of normal and CML CD34+ quiescent (G0) cell, 292 genes were downregulated and 192 genes upregulated in the CML/G0 Cells. The differentially expressed genes were grouped according to their reported functions, and correlations were sought with biological differences previously observed between the same groups. The most relevant findings include the following. (i) CML G0 cells are in a more advanced stage of development and more poised to proliferate than normal G0 cells. (ii) When CML G0 cells are stimulated to proliferate, they differentiate and mature more rapidly than normal counterpart. (iii) Whereas normal G0 cells form only granulocyte/monocyte colonies when stimulated by cytokines, CML G0 cells form a combination of the above and erythroid clusters and colonies. (iv) Prominin-1 is the gene most downregulated in CML G0 cells, and this appears to be associated with the spontaneous formation of erythroid colonies by CML progenitors without EPO.

Abstract The androgen receptor (AR) is the principal target for treatment of non–organ-confined prostate cancer (PCa). Androgen deprivation therapies (ADT) directed against the AR ligand–binding domain do not fully inhibit androgen-dependent signaling critical for PCa progression. Thus, information that could direct the development of more effective ADTs is desired. Systems and bioinformatics approaches suggest that considerable variation exists in the mechanisms by which AR regulates expression of effector genes, pointing to a role for secondary transcription factors. A combination of microarray and in silico analyses led us to identify a 158-gene signature that relies on AR along with the transcription factor SRF (serum response factor), representing less than 6% of androgen-dependent genes. This AR-SRF signature is sufficient to distinguish microdissected benign and malignant prostate samples, and it correlates with the presence of aggressive disease and poor outcome. The AR-SRF signature described here associates more strongly with biochemical failure than other AR target gene signatures of similar size. Furthermore, it is enriched in malignant versus benign prostate tissues, compared with other signatures. To our knowledge, this profile represents the first demonstration of a distinct mechanism of androgen action with clinical relevance in PCa, offering a possible rationale to develop novel and more effective forms of ADT. Cancer Res; 71(5); 1978–88. ©2011 AACR.

Abstract HSulf-1 modulates the sulfation states of heparan sulfate proteoglycans critical for heparin binding growth factor signaling. In the present study, we show that HSulf-1 is transcriptionally deregulated under hypoxia in breast cancer cell lines. Knockdown of HIF-1α rescued HSulf-1 downregulation imposed by hypoxia, both at the RNA and protein levels. Chromatin immunoprecipitation with HIF-1α and HIF-2α antibodies confirmed recruitment of HIF-α proteins to the two functional hypoxia-responsive elements on the native HSulf-1 promoter. HSulf-1 depletion in breast cancer cells resulted in an increased and sustained bFGF2 (basic fibroblast growth factor) signaling and promoted cell migration and invasion under hypoxic conditions. In addition, FGFR2 (fibroblast growth factor receptor 2) depletion in HSulf-1–silenced breast cancer cells attenuated hypoxia-mediated cell invasion. Immunohistochemical analysis of 53 invasive ductal carcinomas and their autologous metastatic lesions revealed an inverse correlation for the expression of HSulf-1 to CAIX in both the primary tumors (P ≥ 0.0198) and metastatic lesions (P ≥ 0.0067), respectively, by χ2 test. Finally, HSulf-1 expression levels in breast tumors by RNA in situ hybridization showed that high HSulf-1 expression is associated with increased disease-free and overall survival (P ≥ 0.03 and P ≥ 0.0001, respectively). Collectively, these results reveal an important link between loss of HSulf-1 under hypoxic microenvironment and increased growth factor signaling, cell migration, and invasion. Cancer Res; 71(6); 2152–61. ©2011 AACR.

Patients with cancer often have complex needs that must be met within a short intervention window. The authors highlight opportunities for improved multidisciplinary care for patients with advanced cancer and their families.

Abstract Estrogen receptors (ERs) play key roles in breast cancer development and influence treatment outcome in breast cancer patients. More than 60% of human breast cancer tumors express ER and many of those are Estrogen (E2) dependent. ER positive tumors are considered to have a good prognosis; however some tumors escape to E2 requirements. Thus, the identification of molecules that regulate ER function may facilitate development of breast cancer treatment strategies. The pro-angiogenic factor CYR61 induces breast cancer cells to bypass the normal estrogen requirements for growth and acquire an estrogen-independent and antiestrogen resistant phenotype. However, the mechanism by which CYR61 regulates the hormonal response in breast cancer is not well understood. We over-expressed CYR61 in the ER positive cells MCF-7 (MCF-7/CYR61). Our results demonstrated that CYR61 induces constitutive transcriptional activity of the ER, which is independent of E2. Moreover, we demonstrated that CYR61 co-localizes with the ER in the nucleus and casually CYR61 transcriptional activity is regulated, at least in part by FOXO3a binding site. In search of the mechanism by which CYR61 that modulates hormonal response we discovered that CYR61 and FOXO3a interact in the presence of E2. This functional interplay among CYR61/ER/FOXO3a most likely plays a critical role in blocking hormone-response of breast cancer growth in vitro and in vivo. Furthermore, these studies demonstrate that CYR61 and FOXO3a are novel therapeutic targets to halt hormone-independent breast carcinomas. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5145.