IU Indianapolis ScholarWorks :: Browsing by Author "Grimes, Brenda R."

Browsing by Author "Grimes, Brenda R."

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“ASSESSMENT OF CHROMOSOME INSTABILITY IN TRIPLE-NEGATIVE BREAST CANCERS USING NUCLEI HARVESTED“ASSESSMENT OF CHROMOSOME INSTABILITY IN TRIPLE-NEGATIVE BREAST CANCERS USING NUCLEI HARVESTED FROM FROZEN TISSUES” FROM FROZEN TISSUES”
(Office of the Vice Chancellor for Research, 2012-04-13) Brown, M. Tony; Slee, Roger B.; Steiner, Camie M.; Radovich, Milan; Schneider, Bryan P.; Grimes, Brenda R.
Chromosomal instability (CIN), defined as ongoing chromosome mis-segregation, is prevalent in the majority of solid tumors and potentially contributes to cancer progression and hazardous genetic changes. Optimization of a common laboratory technique to assess CIN in isolated nuclei will benefit basic research and possibly be useful for clinical diagnostic purposes. Preliminary studies have demonstrated a successful protocol for performing fluorescence in situ hybridization (FISH) on nuclei harvested from frozen tumor and normal breast tissues. The frozen breast tumors were of the triple-negative breast cancer (TNBC) sub-type that does not express estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor-2 (HER2). Six TNBCs analyzed to date (20-50 nuclei per tumor) exhibited chromosome instability using centromere specific probes in FISH analysis. Modal centromere number deviation (MCD)/sample was used to calculate CIN levels. Percent MCD ranged from 32-68% in TNBCs and contrasted with the normal breast tissue sample that exhibited 2% MCD. Previous FISH studies on tissue sections by others have shown that ER negative breast tumors with greater than 45% MCD had a better prognosis. Further study will be required to determine whether CIN levels (measured by MCD) can serve as a biomarker for stratifying TNBC patients into likely responders and non- responders to treatment. Chromatin immunoprecipitation (ChIP) assays performed in parallel from the same frozen tissue revealed that centromeric heterochromatin structure is altered in TNBCs and may contribute to chromosome instability. The ability to perform both FISH and ChIP analysis on frozen human breast tissue has provided a foundation for further exploration of the relationship between CIN and centromere malfunction in tumor tissues and opens up therapeutic possibilities targeting the CIN phenotype in TNBCs.
Consequences of telomerase inhibition and telomere dysfunction in BRCA1 mutant cancer cells
(2014-03-12) Phipps, Elizabeth Ann; Herbert, Brittney-Shea; Grimes, Brenda R.; Sledge, George W.; Turchi, John; White, Kenneth E.
Telomere maintenance is a critical component of genomic stability. An increasing body of evidence suggests BRCA1, a tumor suppressor gene with a variety of functions including DNA repair and cell cycle regulation, plays a role in telomere maintenance. Mutations in BRCA1 account for approximately half of all hereditary breast and ovarian cancers, and the gene is silenced via promoter methylation and loss of heterozygosity in a proportion of sporadic breast and ovarian cancers. The objective of this study was to determine whether GRN163L, a telomerase inhibitor, currently in clinical trials for the treatment of cancer, has enhanced anti-cancer activity in BRCA1 mutant breast/ovarian cancer cell lines compared to wild-type cancer cells. BRCA1 mutant cancer cells were observed to have shorter telomeres and increased sensitivity to telomerase inhibition, compared to cell lines with wild-type BRCA1. Importantly, GRN163L treatment was synergistic with DNA-damaging drugs, suggesting potential synthetic lethality of the BRCA1 cancer subtype and telomerase inhibition In a related study to examine the roles of BRCA1/2 in telomere maintenance, DNA and RNA extracted from peripheral blood were used to investigate the age-adjusted telomere lengths and telomere-related gene expression profiles of BRCA1 and BRCA2 individuals compared to individuals who developed sporadic cancer and healthy controls. BRCA1 mutation carriers and breast cancer patients showed the shortest average telomere lengths compared to the other groups. In addition, distinct genomic profiles of BRCA mutation carriers were obtained regarding overexpression of telomere-related genes compared to individuals who developed sporadic or familial breast cancer. In summary, telomerase inhibition may be a viable treatment option in BRCA1 mutant breast or ovarian cancers. These data also provides insights into further investigations on the role of BRCA1 in the biology underlying telomere dysfunction in cancer development.
DECODING THE TRANSCRIPTIONAL LANDSCAPE OF TRIPLE-NEGATIVE BREAST CANCER USING NEXT GENERATION WHOLE TRANSCRIPTOME SEQUENCING
(2012-03-16) Radovich, Milan; Schneider, Bryan P.; Flockhart, David A.; Ivan, Mircea; Herbert, Brittney-Shea; Grimes, Brenda R.; Nakshatri, Harikrishna
Triple-negative breast cancers (TNBCs) are negative for the expression of estrogen (ER), progesterone (PR), and HER-2 receptors. TNBC accounts for 15% of all breast cancers and results in disproportionally higher mortality compared to ER & HER2-positive tumours. Moreover, there is a paucity of therapies for this subtype of breast cancer resulting primarily from an inadequate understanding of the transcriptional differences that differentiate TNBC from normal breast. To this end, we embarked on a comprehensive examination of the transcriptomes of TNBCs and normal breast tissues using next-generation whole transcriptome sequencing (RNA-Seq). By comparing RNA-seq data from these tissues, we report the presence of differentially expressed coding and non-coding genes, novel transcribed regions, and mutations not previously reported in breast cancer. From these data we have identified two major themes. First, BRCA1 mutations are well known to be associated with development of TNBC. From these data we have identified many genes that work in concert with BRCA1 that are dysregulated suggesting a role of BRCA1 associated genes with sporadic TNBC. In addition, we observe a mutational profile in genes also associated with BRCA1 and DNA repair that lend more evidence to its role. Second, we demonstrate that using microdissected normal epithelium maybe an optimal comparator when searching for novel therapeutic targets for TNBC. Previous studies have used other controls such as reduction mammoplasties, adjacent normal tissue, or other breast cancer subtypes, which may be sub-optimal and have lead to identifying ineffective therapeutic targets. Our data suggests that the comparison of microdissected ductal epithelium to TNBC can identify potential therapeutic targets that may lead to be better clinical efficacy. In summation, with these data, we provide a detailed transcriptional landscape of TNBC and normal breast that we believe will lead to a better understanding of this complex disease.
An inhibitor of the mitotic kinase, MPS1, is selective towards pancreatic cancer cells
(Office of the Vice Chancellor for Research, 2014-04-11) Bansal, Ruchi; Blackburn, Corrine; Brown, Lyndsey; Gasaway, Rachel; Victorino, Jose; Jeong, Jaesik; Gore, Jesse; March, Keith L.; Herbert, Brittney-Shea; Colombo, Riccardo; Korc, Murray; Slee, Roger B.; Grimes, Brenda R.
The abysmal five year pancreatic cancer survival rate of less than 6% highlights the need for new treatments for this deadly malignancy. Cytotoxic drugs normally target rapidly dividing cancer cells but unfortunately often target stem cells resulting in toxicity. This warrants the development of compounds that selectively target tumor cells. An inhibitor of the mitotic kinase, MPS1, which has been shown to be more selective towards cancer cells than non-tumorigenic cells, shows promise but its effects on stem cells has not been investigated. MPS1 is an essential component of the Spindle Assembly Checkpoint and is proposed to be up-regulated in cancer cells to maintain chromosomal segregation errors within survivable limits. Inhibition of MPS1 kinase causes cancer cell death accompanied by massive aneuploidy. Our studies demonstrate that human adipose stem cells (ASCs) and can tolerate higher levels of a small molecule MPS1 inhibitor than pancreatic cancer cells. In contrast to PANC-1 cancer cells, ASCs and telomerase-immortalized pancreatic ductal epithelial cells did not exhibit elevated chromosome mis-segregation after treatment with the MPS1 inhibitor for 72hrs. In contrast, PANC-1 pancreatic cancer cells exhibited a large increase in chromosomal mis-segregation under similar conditions. Furthermore, growth of ASCs was minimally affected post treatment whereas PANC-1 cells were severely growth impaired suggesting a favorable therapeutic index. Our studies, demonstrate that MPS1 inhibition is selective towards pancreatic cancer cells and that stem cells are less affected in vitro. These data suggest MPS1 inhibition should be further investigated as a new treatment approach in pancreatic cancer.
An inhibitor of the mitotic kinase, MPS1, is selective towards pancreatic cancer cells
(2014) Bansal, Ruchi; Grimes, Brenda R.; Herbert, Brittney-Shea; Dlouhy, Stephen Robert
The abysmal five year pancreatic cancer survival rate of less than 6% highlights the need for new treatments for this deadly malignancy. Cytotoxic drugs normally target rapidly dividing cancer cells but unfortunately often target stem cells resulting in toxicity. This warrants the development of compounds that selectively target tumor cells. An inhibitor of the mitotic kinase, MPS1, which has been shown to be more selective towards cancer cells than non-tumorigenic cells, shows promise but its effects on stem cells has not been investigated. MPS1 is an essential component of the Spindle Assembly Checkpoint and is proposed to be up-regulated in cancer cells to maintain chromosomal segregation errors within survivable limits. Inhibition of MPS1 kinase causes cancer cell death accompanied by massive aneuploidy. Our studies demonstrate that human adipose stem cells (ASCs) and can tolerate higher levels of a small molecule MPS1 inhibitor than pancreatic cancer cells. In contrast to PANC-1 cancer cells, ASCs and telomerase-immortalized pancreatic ductal epithelial cells did not exhibit elevated chromosome mis-segregation after treatment with the MPS1 inhibitor for 72hrs. In contrast, PANC-1 pancreatic cancer cells exhibited a large increase in chromosomal mis-segregation under similar conditions. Furthermore, growth of ASCs was minimally affected post treatment whereas PANC-1 cells were severely growth impaired suggesting a favorable therapeutic index. Our studies, demonstrate that MPS1 inhibition is selective towards pancreatic cancer cells and that stem cells are less affected in vitro. These data suggest MPS1 inhibition should be further investigated as a new treatment approach in pancreatic cancer.
Input DNA Ratio Determines Copy Number of The 33 kb Factor IX Gene on De Novo Human Artificial Chromosomes
(Elsevier, 2007-12-04) Breman, Amy M.; Steiner, Camie M.; Slee, Roger B.; Grimes, Brenda R.; Medical and Molecular Genetics, School of Medicine
Human artificial chromosomes (ACs) are non-integrating vectors that may be useful for gene therapy. They assemble in cultured cells following transfection of human centromeric α -satellite DNA and segregate efficiently alongside the host genome. In the present study, a 33 kilobase (kb) Factor IX (FIX) gene was incorporated into mitotically stable ACs in human HT1080 lung derived cells using co-transfection of a bacterial artificial chromosome (BAC) harboring synthetic α -satellite DNA and a P1 artificial chromosome(PAC) that spans the FIX locus. ACs were detected in ≥90% of chromosome spreads in 8 of 19 lines expanded from drug resistant colonies. FIX transgene copy number on ACs was determined by input DNA transfection ratios. Furthermore, a low level of FIX transcription was detected from ACs with multiple transgenes but not from those incorporating a single transgene, suggesting that reducing transgene number may limit misexpression. Their potential to segregate cross species was measured by transferring ACs into mouse and hamster cell lines using microcell-mediated chromosome transfer. Lines were obtained where ACs segregated efficiently. The stable segregation of ACs in rodent cells suggests that it should be possible to develop animal models to test the capacity of ACs to rescue FIX deficiency.
Phenotypic plasticity in normal breast derived epithelial cells
(Biomed Central, 2014) Sauder, Candice A. M.; Koziel, Jillian E.; Choi, MiRan; Fox, Melanie J.; Grimes, Brenda R.; Badve, Sunil S.; Blosser, Rachel J.; Radovich, Milan; Lam, Christina C.; Vaughan, Melville B.; Herbert, Brittney-Shea; Clare, Susan E.; Pathology and Laboratory Medicine, School of Medicine
Background Normal, healthy human breast tissue from a variety of volunteer donors has become available for research thanks to the establishment of the Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center (KTB). Multiple epithelial (K-HME) and stromal cells (K-HMS) were established from the donated tissue. Explant culture was utilized to isolate the cells from pieces of breast tissue. Selective media and trypsinization were employed to select either epithelial cells or stromal cells. The primary, non-transformed epithelial cells, the focus of this study, were characterized by immunohistochemistry, flow cytometry, and in vitro cell culture. Results All of the primary, non-transformed epithelial cells tested have the ability to differentiate in vitro into a variety of cell types when plated in or on biologic matrices. Cells identified include stratified squamous epithelial, osteoclasts, chondrocytes, adipocytes, neural progenitors/neurons, immature muscle and melanocytes. The cells also express markers of embryonic stem cells. Conclusions The cell culture conditions employed select an epithelial cell that is pluri/multipotent. The plasticity of the epithelial cells developed mimics that seen in metaplastic carcinoma of the breast (MCB), a subtype of triple negative breast cancer; and may provide clues to the origin of this particularly aggressive type of breast cancer. The KTB is a unique biorepository, and the normal breast epithelial cells isolated from donated tissue have significant potential as new research tools.
A portable BRCA1-HAC (human artificial chromosome) module for analysis of BRCA1 tumor suppressor function
(Oxford University Press, 2014-12-01) Kononenko, Artem V.; Bansal, Ruchi; Lee, Nicholas C.O.; Grimes, Brenda R.; Masumoto, Hiroshi; Earnshaw, William C.; Larionov, Vladimir; Kouprina, Natalay; Department of Medical & Molecular Genetics, IU School of Medicine
BRCA1 is involved in many disparate cellular functions, including DNA damage repair, cell-cycle checkpoint activation, gene transcriptional regulation, DNA replication, centrosome function and others. The majority of evidence strongly favors the maintenance of genomic integrity as a principal tumor suppressor activity of BRCA1. At the same time some functional aspects of BRCA1 are not fully understood. Here, a HAC (human artificial chromosome) module with a regulated centromere was constructed for delivery and expression of the 90 kb genomic copy of the BRCA1 gene into BRCA1-deficient human cells. A battery of functional tests was carried out to demonstrate functionality of the exogenous BRCA1. In separate experiments, we investigated the role of BRCA1 in maintenance of heterochromatin integrity within a human functional kinetochore. We demonstrated that BRCA1 deficiency results in a specific activation of transcription of higher-order alpha-satellite repeats (HORs) assembled into heterochromatin domains flanking the kinetochore. At the same time no detectable elevation of transcription was observed within HORs assembled into centrochromatin domains. Thus, we demonstrated a link between BRCA1 deficiency and kinetochore dysfunction and extended previous observations that BRCA1 is required to silence transcription in heterochromatin in specific genomic loci. This supports the hypothesis that epigenetic alterations of the kinetochore initiated in the absence of BRCA1 may contribute to cellular transformation.
Selective inhibition of pancreatic ductal adenocarcinoma cell growth by the mitotic MPS1 kinase inhibitor NMS-P715
(American Association for Cancer Research, 2014-02) Slee, Roger B.; Grimes, Brenda R.; Bansal, Ruchi; Gore, Jesse; Blackburn, Corinne; Brown, Lyndsey; Gasaway, Rachel; Jeong, Jaesik; Victorino, Jose; March, Keith L.; Colombo, Riccardo; Herbert, Brittney-Shea; Korc, Murray; Department of Medical and Molecular Genetics, IU School of Medicine
Most solid tumors, including pancreatic ductal adenocarcinoma (PDAC), exhibit structural and numerical chromosome instability (CIN). Although often implicated as a driver of tumor progression and drug resistance, CIN also reduces cell fitness and poses a vulnerability that can be exploited therapeutically. The spindle assembly checkpoint (SAC) ensures correct chromosome-microtubule attachment, thereby minimizing chromosome segregation errors. Many tumors exhibit upregulation of SAC components such as MPS1, which may help contain CIN within survivable limits. Prior studies showed that MPS1 inhibition with the small molecule NMS-P715 limits tumor growth in xenograft models. In cancer cell lines, NMS-P715 causes cell death associated with impaired SAC function and increased chromosome missegregation. Although normal cells appeared more resistant, effects on stem cells, which are the dose-limiting toxicity of most chemotherapeutics, were not examined. Elevated expression of 70 genes (CIN70), including MPS1, provides a surrogate measure of CIN and predicts poor patient survival in multiple tumor types. Our new findings show that the degree of CIN70 upregulation varies considerably among PDAC tumors, with higher CIN70 gene expression predictive of poor outcome. We identified a 25 gene subset (PDAC CIN25) whose overexpression was most strongly correlated with poor survival and included MPS1. In vitro, growth of human and murine PDAC cells is inhibited by NMS-P715 treatment, whereas adipose-derived human mesenchymal stem cells are relatively resistant and maintain chromosome stability upon exposure to NMS-P715. These studies suggest that NMS-P715 could have a favorable therapeutic index and warrant further investigation of MPS1 inhibition as a new PDAC treatment strategy.

Browsing by Author "Grimes, Brenda R."

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