Isolation of Genomic DNA from Mammalian Cells and Fixed Tissue.

Examination of DNA variation is central to understanding the function of mammalian cells, tissues, and whole bodies. Extraction of high-quality DNA from cells and tissues is necessary for innumerable different experiments. We present protocols for the extraction of DNA from both fresh samples and formalin-fixed tissue. Methods for extracting DNA have been standardized and streamlined over the past couple of decades and many extraction kits are available for a reasonable cost. In addition, many of the extraction procedures can also be automated for even higher throughput sample preparation. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: DNA isolation from whole blood, tissue, and cultured cells Alternate Protocol: DNA extraction using automated machines Basic Protocol 2: DNA isolation from saliva and buccal swabs Basic Protocol 3: DNA isolation from formalin-fixed, paraffin-embedded tissue.

NF-κB activation-induced anti-apoptosis renders HER2-positive cells drug resistant and accelerates tumor growth.

UNLABELLED: Breast cancers with HER2 overexpression are sensitive to drugs targeting the receptor or its kinase activity. HER2-targeting drugs are initially effective against HER2-positive breast cancer, but resistance inevitably occurs. We previously found that NF-κB is hyperactivated in a subset of HER2-positive breast cancer cells and tissue specimens. In this study, we report that constitutively active NF-κB rendered HER2-positive cancer cells resistant to anti-HER2 drugs and cells selected for lapatinib resistance upregulated NF-κB. In both circumstances, cells were antiapoptotic and grew rapidly as xenografts. Lapatinib-resistant cells were refractory to HER2 and NF-κB inhibitors alone but were sensitive to their combination, suggesting a novel therapeutic strategy. A subset of NF-κB-responsive genes was overexpressed in HER2-positive and triple-negative breast cancers, and patients with this NF-κB signature had poor clinical outcome. Anti-HER2 drug resistance may be a consequence of NF-κB activation, and selection for resistance results in NF-κB activation, suggesting that this transcription factor is central to oncogenesis and drug resistance. Clinically, the combined targeting of HER2 and NF-κB suggests a potential treatment paradigm for patients who relapse after anti-HER2 therapy. Patients with these cancers may be treated by simultaneously suppressing HER2 signaling and NF-κB activation. IMPLICATIONS: The combination of an inhibitor of IκB kinase (IKK) inhibitor and anti-HER2 drugs may be a novel treatment strategy for drug-resistant human breast cancers.

PALB2 mutations in familial breast and pancreatic cancer.

PALB2 (Partner And Localizer of BRCA2) binds to and co-localizes with BRCA2 in DNA repair. Germline mutations in PALB2 have been identified in approximately 1-2% of familial breast cancer and 3-4% of familial pancreatic cancer cases. The goal of this study was to evaluate the prevalence of PALB2 mutations in women with breast cancer without BRCA1/2 mutations who also had a personal or family history of pancreatic cancer. PALB2 mutation analysis was performed in 94 non-BRCA1/2 breast cancer patients with a personal or family history of pancreatic cancer. Two truncating PALB2 mutations, c.3549C>CA and c.2962C>CT, were identified resulting in a mutation prevalence of 2.1%. The proband found to carry the c.3549C>CA PALB2 mutation had a mother diagnosed with both breast and pancreatic cancer; this relative was subsequently confirmed to carry the identical mutation. The proband with the c.2962C>CT mutation had a father and paternal aunt diagnosed with pancreatic cancer; neither relative was available for testing. Two novel PALB2 missense variants were also found, one of which was deemed potentially deleterious. The prevalence rate of PALB2 mutations in a non-BRCA1/2 breast cancer population specifically selected for a family history of pancreatic cancer does not appear to be significantly increased compared to that observed in other breast cancer populations studied thus far. Further evaluation is needed to determine the prevalence of PALB2 mutations and the clinical utility of such testing in those individuals affected with both breast and pancreatic cancers.

Serial transplantation of NMU-induced rat mammary tumors: a model of human breast cancer progression.

Human breast cancer is a heterogeneous disease that appears to progress from an in situ tumor to invasive cancer. Little is known about the molecular events driving this progression. Although microarray technology has helped us understand the genetic heterogeneity of breast cancer, its application to studying the transition from in situ to invasive disease is limited by the inability to follow the progression of a single patient's tumor. We previously used rat specific microarrays to show that N-methyl-N-nitrosourea induced tumors are similar to low-grade estrogen-receptor positive human breast cancer. Here, we transplanted these tumors through 5 generations of syngeneic hosts, and studied 65 resulting tumors. Most transplanted tumors gradually progressed from a noninvasive, low-grade cancer to a higher-grade invasive disease, losing p63 localization and basement membrane integrity. Invasive cancers frequently demonstrated a more mesenchymal phenotype with increased vimentin expression. Additionally, a unique transplant series is described with a phenotype similar to human basal-like breast cancer. Rat-specific Affymetrix gene arrays containing 15,866 gene probes identified genes that differentiated highly invasive tumors from those of low invasive potential. A linear regression analysis was used to find genes whose change in expression paralleled increasing invasive features independent of the transplant lineage of origin. Genes identified were assigned membership in cell adhesion, signal transduction, cell cycle and extracellular matrix groups, among others. This animal model overcomes the difficulty in studying human breast cancer progression. Our data support a gradual and continuous alteration in programs of gene expression during breast cancer invasion.

Gene expression profiling of NMU-induced rat mammary tumors: cross species comparison with human breast cancer.

Breast cancer is a complex genetic disease characterized by the accumulation of multiple molecular alterations. The NMU breast cancer model induced in the rat is used for the study of mammary carcinogenesis because it closely mimics human breast disease. To assess the validity of this model from a more global molecular perspective, and also to devise a general technique to compare animal profiles with human microarray studies, we have characterized 25 NMU-induced mammary tumors and 11 normal glands using a combination of immunohistochemical and microarray analyses. The rat mammary carcinomas were classified as non-invasive, ER-positive ductal carcinomas with a composition of differentiated epithelial and myoepithelial cell lineages. Gene expression profiles generated using rat Affymetrix arrays containing 15,866 genes demonstrated that the rat mammary tumors are homogeneous and that H-ras mutations did not confer a unique molecular signature. We compared the resulting rat profiles with those obtained from a human dataset by merging the raw microarray data, using an approach that involves a combination of cross-species and cross-platform analysis. Using this novel strategy, we demonstrate the ability of 2305 rat orthologs to recapitulate the classification of human tumors derived from human Affymetrix arrays. The gene expression profiles of the NMU-induced primary tumors were most similar to ER-positive, low to intermediate grade breast cancer. Our technique provides a means to correlate gene expression data from animal models of cancer to human cancer and disease states.