Validation of Driver Mutations最新文献

{"title":"Abstract B47: Validation of novel breast cancer drivers using mammary stem cell-based somatic mouse models","authors":"Zheng Zhang, J. Christin, Chunhui Wang, K. Ge, M. Oktay, Wenjun Guo","doi":"10.1158/1557-3125.ADVBC17-B47","DOIUrl":"https://doi.org/10.1158/1557-3125.ADVBC17-B47","url":null,"abstract":"Cancer genomics has provided an unprecedented opportunity for understanding genetic causes of human cancer. However, distinguishing which mutations are functionally relevant to cancer pathogenesis remains a major challenge. We describe here a mammary stem cell (MaSC) organoid-based approach for rapid generation of somatic genetically engineered mouse models (GEMMs). By using RNAi and CRISPR-mediated genome engineering in MaSC-GEMMs, we have discovered that inactivation of Ptpn22 or Mll3, two genes mutated in human breast cancer, greatly accelerated PI3K-driven mammary tumorigenesis. Using these tumor models, we have also identified genetic alterations promoting tumor metastasis and causing resistance to PI3K- targeted therapy. Both Ptpn22 and Mll3 inactivation resulted in disruption of mammary gland differentiation and an increase in stem cell activity. Mechanistically, Mll3 deletion enhanced stem cell activity through activation of the HIF pathway. Thus, our study has established a robust in vivo platform for functional cancer genomics and has discovered functional breast cancer mutations. Citation Format: Zheng Zhang, John R. Christin, Chunhui Wang, Kai Ge, Maja Oktay, Wenjun Guo. Validation of novel breast cancer drivers using mammary stem cell-based somatic mouse models [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B47.","PeriodicalId":175334,"journal":{"name":"Validation of Driver Mutations","volume":"1 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2018-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"130021524","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B42: Identification of CPT1A as a novel driver of proliferation in luminal breast cancer","authors":"Kristina Mastrioanni, Kimberly A. Parker, Michael L. Gatza","doi":"10.1158/1557-3125.ADVBC17-B42","DOIUrl":"https://doi.org/10.1158/1557-3125.ADVBC17-B42","url":null,"abstract":"Clinically, approximately two-thirds of the nearly 250,000 breast cancer cases diagnosed each year in the United States are hormone receptor-positive, luminal breast tumors. Slower-growing luminal breast tumors are often successfully treated using endocrine-based therapies, resulting in a relatively good prognosis for these patients. However, more highly proliferative luminal tumors, even with the recent approval of CDK4/6 inhibitors, are often resistant, or become resistant, to current therapies, leading to a worse outcome for these women. Therefore, it is necessary to identify genetic events responsible for tumorigenesis and to identify potential druggable genetic alterations and/or pathways in order to improve clinical outcome. To address this question, we recently developed and used an innovative genomics-based strategy to interrogate orthogonal genome-wide data from more than 2,500 patients from the TCGA and METABRIC studies. By using an 11-gene mRNA-based gene expression signature of proliferation as a conceptual framework, we identified DNA copy number alterations and somatic mutations associated with luminal breast cancer proliferation. In order to identify the subset of amplified genes that are essential for cell viability, we analyzed a dataset of 27 breast cancer cell lines with available mRNA expression and genome-wide shRNA proliferation data. By integrating DNA copy number and shRNA analyses, we identified amplified carnitine palmitoyltransferase 1A (CPT1A) as a novel driver of proliferation in luminal breast cancer. CPT1A was found to be amplified in 40.2% of highly proliferative (top quartile) luminal tumors compared to 24.7% of all other samples and CPT1A DNA copy number status correlated with mRNA expression levels. Importantly, CPT1A was shown to be essential in luminal breast cancer cell lines with a high proliferation signature score. By analyzing RPPA data from luminal breast tumors, we confirmed CPT1A amplified tumors have increased expression of protein markers of proliferation. Since CPT1A is the rate-limiting enzyme responsible for fatty acid import into the mitochondria during fatty acid β oxidation (FAO), these data indicate that highly proliferative luminal tumors may utilize FAO as a prominent energy source. Interestingly, our analyses of RNA-seq data (n=1,031) demonstrated that highly proliferative luminal tumors have significantly higher CPT1A mRNA expression than either basal-like, which are largely synonymous with TNBC, or less proliferative luminal tumors, suggesting that these tumors may have a greater dependency on CPT1A activity. CPT1A has two major variants: cytoplasmic Variant 1 is the rate-limiting enzyme responsible for fatty acid import into the mitochondria during FAO while nuclear Variant 2 is purported to mediate histone deacetylase activity. Analyses of RNAseq data (n=819) demonstrated that Variant 1 represents >99.2% of CPT1A mRNA transcripts in a given tumor, suggesting the FAO-associated role of C","PeriodicalId":175334,"journal":{"name":"Validation of Driver Mutations","volume":"47 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2018-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"116316436","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B43: NF1 deficiency induces aggressive mammary carcinomas in a CRISPR rat model and correlates with poor outcome in sporadic human breast cancer","authors":"Patrick S. Dischinger, Elizabeth A. Tovar, Curt J. Essenburg, E. Gardner, Megan E. Callaghan, M. Bowman, Z. Madaj, Ashley N. Turner, A. Challa, Tristan Kempston, B. Eagleson, R. Bronson, R. Kesterson, Matthew R. Steensma, Carrie Graveel","doi":"10.1158/1557-3125.ADVBC17-B43","DOIUrl":"https://doi.org/10.1158/1557-3125.ADVBC17-B43","url":null,"abstract":"Neurofibromatosis type 1 (NF1) is a common tumor predisposition syndrome caused by germline mutations in the NF1 gene that affects1 in 3,000 births. The NF1 gene encodes for neurofibromin, a GTPase that negatively regulates RAS. Women with NF1 have a 10-fold increased risk of developing breast cancer under the age of 40 and an overall lifetime risk of 18%. NF1-related breast cancers are associated with higher grade, poor prognostic factors, and poor survival (5-year survival is 68%). Comprehensive genomic analyses have also revealed that NF1 is commonly mutated in sporadic breast cancers and is a suspected driver gene. Much of our understanding of the mechanisms underlying the functional loss of NF1 comes from genetically engineered mouse models that do not completely recapitulate the phenotypes of human NF1 . To examine the effect of functional NF1 loss on tumorigenesis, we induced a deletion in the NF1 GAP-related domain (GRD) in Sprague-Dawley rats using a CRISPR-Cas9 editing strategy. Because CRISPR modification results in variability at the guide site, two classes of Nf1- deficient rats were generated: larger “in-frame” indels (i.e., 63 bp) and smaller “premature stop” indels (i.e., -11). Both types of Nf1 indels induced highly penetrant and aggressive mammary adenocarcinomas in multiple rat founder lines. Mammary tumors were observed in 100% (11/11) of G0 females in mammary glands 1-5 at the average age of 51 days. Notably, two male founders also developed mammary adenocarcinomas at 14-16 months of age. This aggressive mammary phenotype was highly penetrant and multifocal mammary tumors were observed in the subsequent F1 and F2 generations from each of the Nf1 lines. Histopathologic analysis of the mammary tumors revealed that both Nf1 in-frame deletions and premature stop indels induced a wide variety of histopathologic mammary tumor types (i.e., acinar, solid, ductular, and cystic). To understand how the distinct Nf1 indels affect disease burden and survival, we examined the overall survival of females with in-frame indels (n=35) compared to premature stop indels (n=24) in three lines ( Nf1 IF-57/+ , Nf1 IF-57/PS-8 , Nf1 IF-54/PS 11 ). Kaplan-Meier analysis revealed that animals with a premature stop codon in exon 20 died due to tumor burden significantly faster than animals with an in-frame deletion (p Nf1 in-frame deletions may maintain some functional activity (compared to premature stop indels) that delays tumorigenesis. To our knowledge, this is the first model of NF1-related breast cancer and one of the few animal models of male breast cancer. Moreover, the Nf1 CRISPR rat is a novel model in which to explore the role of NF1 and deregulated Ras signaling in breast cancer initiation and progression. To examine the frequency of NF1 alterations in sporadic human breast cancer, we analyzed the METABRIC breast cancer dataset. NF1 mutations were identified in 3.8% of patients, whereas 24.5% of cancers harbored shallow deletions in NF1 . Su","PeriodicalId":175334,"journal":{"name":"Validation of Driver Mutations","volume":"8 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2018-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"117099079","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B41: BP1 is an important biomarker in breast cancer","authors":"P. Berg, Y. Man, S. Simmens, S. Fu, L. Cavalli, F. Vesuna, S. Kirolikar, A. Schwartz","doi":"10.1158/1557-3125.ADVBC17-B41","DOIUrl":"https://doi.org/10.1158/1557-3125.ADVBC17-B41","url":null,"abstract":"Beta protein 1 (BP1), a transcription factor (TF) we identified and cloned, is encoded by a homeobox gene called DLX4. BP1 is overexpressed in breast cancer, prostate cancer, ovarian cancer, acute myeloid leukemia, non-small cell lung cancer, and possibly other malignancies as well. Important characteristics of BP1 in breast cancer, a focus in our laboratory, include findings that: (1) BP1 is expressed in 80% of invasive ductal breast tumors, including 89% of the tumors of African American women. These data are based on both RNA and protein data. (2) BP1 expression correlates with the progression of breast tumors, from 0% in normal breast tissue to 21% in hyperplasia and 46% in ductal carcinoma in situ. (3) BP1, which maps to 17q21, can be activated by DNA amplification. (4) BP1 appears to be associated with metastasis. Forty-six cases of inflammatory breast cancer were examined; all were positive for BP1 expression. Nine cases had metastasized; lymph nodes from all nine were also BP1 positive. Moreover, BP1 activates a known trigger of metastasis, the Twist gene. (5) BP1 overexpression induces oncogene expression. BP1 activates the BCL-2 gene; high BCL-2 protein levels are associated with resistance to drug and radiation therapy. BP1 also activates VEGF and c-MYC, as well as other genes important in angiogenesis, invasion, and metastasis. (6) Cells overexpressing BP1, when injected into the fat pads of nude mice, were associated with larger and more frequent tumors than found in control mice receiving cells with low BP1. In summary, BP1 appears to confer properties on breast cancer cells that lead to a more invasive and aggressive phenotype. Since the functions of homeotic transcription factors are highly conserved, it is possible that BP1 regulates many of the same processes and genes in other malignancies in which it is active. Citation Format: Patricia E. Berg, Yan-gao Man, Samuels Simmens, Sidney W. Fu, Lucianne Cavalli, Farhad Vesuna, Saurabh Kirolikar, Arnold Schwartz. BP1 is an important biomarker in breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B41.","PeriodicalId":175334,"journal":{"name":"Validation of Driver Mutations","volume":"21 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2018-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"133699366","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B44: Role of EGFR/ERBB4 and MAPK signaling in modulating oncogenic potential of ER+ breast cancer cells overexpressing HER3 mutant","authors":"Rosalin Mishra, Long Yuan, Thomas Solomon, Samar Alanazi, J. Garrett","doi":"10.1158/1557-3125.ADVBC17-B44","DOIUrl":"https://doi.org/10.1158/1557-3125.ADVBC17-B44","url":null,"abstract":"We sought to determine if patient-identified mutations in HER3 enhance ER-mediated transformation in human mammary epithelial cells (MCF-7 and T47D). Furthermore, we examined if HER3 mutations confer resistance to ER-specific inhibitors (fulvestrant and 4-hydroxytamoxifen) in ER+ breast cancer cells. A series of HER3 mutations identified in patient breast tumors (F94L, G284R, D297Y, D313H, K329T, T355I, L792V, and E1261A) were introduced and stable cell lines were generated in ER+ T47D and MCF-7 cells using lentiviral transduction. We identified the HER3 T355I mutant that had significantly higher cell proliferation than wild-type (wt) HER3 in ER+ MCF-7 and T47-D cells using several cell-based assays. We sought to determine whether this oncogenic T355I mutant renders resistance to antiestrogen therapy. These mutations counteracted the effect of the ER inhibitor 4-hydroxytamoxifen but not fulvestrant, indicating a possible role in antiestrogen therapy. ER+ cells overexpressing T355I have increased p-HER3 and p-ERK1/2 expression compared to wt or empty vector (ev) control. However, there was no major change in Akt signaling in HER3 T355I mutant as compared to control. Phospho-RTK array results indicate that ER+ T47D T355I whole-cell lysate had increased p-ERBB4 and ER+ MCF-7 T355I cells had enhanced p-EGFR expression, suggesting possible downstream activation of MAPK signaling via two distinct activation routes in ER+ T47D and MCF-7 cells. ER+ MCF-7 and T47D cells overexpressing T355I HER3 mutant were subjected to the ERBB family inhibitor lapatinib in presence or absence of ER inhibitor fulvestrant and specific ERK1/2 inhibitor, SCH772984. The data indicated that combined treatment of lapatinib and fulvestrant had reduced cell proliferation as compared to individual treatment groups both in ER+ cells overexpressing wt and HER3 T355I mutant. We also observed a significant reduction in cell proliferation when these cells were subjected to co-treatment of lapatinib and SCH772984 as compared to individual treatments. These data indicate that induced transforming activity observed in HER3 T355I mutant is via ERBB4/MAPK pathway in T47D cells and EGFR/MAPK in MCF-7 cells. Similar results were obtained in 3D-Matrigel assay under above treatment conditions. The Western blot data indicated that Cyclin D1 and p-ERK1/2 expression is altered in response to combined treatment of lapatinib with or without fulvestrant and SCH772984 in ER+ T47D and MCF-7 cells, indicating that Cyclin D1 mediates signaling downstream of the MAPK pathway. However, Akt signaling is not affected when cells were treated with EGFR family and ERK1/2 inhibitors or combination of both. We also observed ERα expression is upregulated in ER+ T47D and MCF-7 cells overexpressing HER3 mutant. Experiments are ongoing to determine possible crosstalk between HER3 and ER signaling in ER+ cells overexpressing HER3 mutant. Additionally, we tested the effect of knocking down HER3 in cell lines harboring","PeriodicalId":175334,"journal":{"name":"Validation of Driver Mutations","volume":"17 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2018-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"116738135","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B46: Targeting EZH2 reactivates a breast cancer subtype-specific antimetastatic transcriptional program","authors":"Alison Hirukawa, Harvey W. Smith, Dongmei Zou, P. Savage, Radia M. Johnson, G. Bourque, V. Giguère, M. Basik, C. Dufour, Morag Park, W. Muller","doi":"10.1158/1557-3125.ADVBC17-B46","DOIUrl":"https://doi.org/10.1158/1557-3125.ADVBC17-B46","url":null,"abstract":"Emerging evidence has illustrated the importance of epigenomic reprogramming in cancer, with altered post-translational modifications of histones contributing to the pathogenesis of many tumor types, including breast cancer. However, the contributions of histone modifiers to breast cancer progression are unclear, and how these processes vary between molecular subtypes has yet to be adequately addressed. In this study, we demonstrate that genetic or pharmacologic targeting of the epigenetic modifier Ezh2 dramatically hinders metastatic behavior in a mouse model of breast cancer reflective of the luminal B subtype. We also employ patient-derived xenografts from different intrinsic subtypes of breast cancer to confirm that pharmacologic inhibition of Ezh2 activity hinders metastasis specifically in the luminal B subtype. We further define a molecular mechanism intrinsic to the luminal B subtype whereby EZH2 maintains H3K27me3-mediated repression of the FOXC1 gene, thereby inactivating a FOXC1-driven, antiinvasive transcriptional program. We demonstrate that higher FOXC1 levels are predictive of favorable outcome specifically in luminal B breast cancer patients and establish the use of EZH2 methyltransferase inhibitors as a viable strategy to block metastasis in luminal B breast cancer, where options for targeted therapy are currently limited. Citation Format: Alison Hirukawa, Harvey Smith, Dongmei Zou, Paul Savage, Radia Johnson, Guillaume Bourque, Vincent Giguere, Mark Basik, Cathy Dufour, Morag Park, William Muller. Targeting EZH2 reactivates a breast cancer subtype-specific antimetastatic transcriptional program [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B46.","PeriodicalId":175334,"journal":{"name":"Validation of Driver Mutations","volume":"118 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2018-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"132027120","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}