1 BRCA1 in Breast Cancer Elizabeth Pittman

2 Mutation in chromosome 17 linked to hereditary breast cancer1990- Using pairs first-third degree relatives, shared alleles were counted and identified alleles of similar size by southern blot; probes to markers on chromosome 17q21; compared fragments and found 30 different alleles (fragments were different lengths among unrelated subjects) → determined D17S74 is linked to breast cancer (lod score sum = +3.28), then looked at early onset of cancer gene was in this region of 600 kb, identified a transcription unit in center at D17S855 Breast cancer susceptibility gene Miki et al. (1994) Hall et al. (1990)

3 Caestecker and Van de Walle. (2012)BRCA1 functions with tumor suppressors and transcription factors in response to DNA damage Has several functions, but role as tumor suppressor is dependent on role in homologous recombination. Other functions: cell cycle checkpoint, ubiquitination, apoptosis When DNA is damaged, complex of Mre11, RAD50, and Nbs1 (together MRN) bind the broken double stranded DNA and recruit ATM (ataxia telegiectasia mutated), a DNA damage sensor. ATM phosphorylates H2AX ATR phosphorylates H2AX in normal DNA replication Directly on SQ motif or indirectly by phosporylating Chk2, which phosphorylates BRCA1 at SQ domain (ser 988) Chk1 can activate BRCA1 by binding to BRCT domain Caestecker and Van de Walle. (2012)

4 Mutations in Ser988 are responsible for loss of homologous recombinationOver 1800 mutations found (not all in cancer), large gene Ring domain - mutations C61G and C64G, protein is less stable because its now more easily degraded & loses its function of E3 ubiquitin ligase because BARD1 cannot bind H2AX marker for DNA damage SQ cluster region - site in DNA damage response proteins, for ATM/ATR phosphorylation S988A mut = normal S phase checkpoint but defective homologous recombination in DNA repair Serine 998 controls homologous recombination and inhibits end joining (not a good method of repair) S1423A mut = normal DNA repair but G2 checkpoint is impaired Mice: Knock in S971 (= to S988 in humans) and ionizing radiation→ mammary and epithelial tumors; homologous recombination is affected but checkpoint is not Mutations in BRCT domain affect localization to damaged strands Zhang and Powell. (2005)

5 BRCA1 required to localize PALB2, BRCA2, RAD51 to repair DNA damageScully et al. (1997) Fan Zhang et al. (2009) Due to its role in cellular proliferation, BRCA1 was thought to be a nuclear protein. Rad51 was known to act at DNA breaks for HR. Co-localized BRCA1 and RAD51 in the same place in the cell. Immunostained with monoclonal antibody for BRCA1 (green) and an affinity purified rabbit polyclonal antiserum for RAD51 (red). Where the two overlap = yellow stain. A-C cells growth not in sync, D-E cells serum starved, in S phase. A. Found another breast cancer susceptibility gene, didn’t know how or if it interacted with BRCA1, but knew it did PALB2. Co-localized the 3 with and without BRCA1- red fluorescent protein with antibodies, green to H2AX (marker that shows DNA has been damaged) Left - HCC1937 has truncated BRCA1 with no C-terminus, so it cant localize to DNA damage. Right – WT BRCA added, co-localization shown by yellow fluorescence  also shown on chart: PALB2 BRCA2 foci are not present at damage site without BRCA1. B. siRNA to knock down BRCA1 expression- complementary strand of ss siRNA binds to target strand and silences it. Immunoblot for protein expression C. Shows siRNA (knocking down BRCA1) affects foci formation of PARB2 and BRCA2 Feng Zhang et al. (2009)

6 BRCA1 knockout mouse models support its role in tumor suppression in mammary tissueIn mice, double mutants are embryonic lethal 5-13 days; BRCA1 required for neural tube development; targeted exon 11 for deletion, lethal at days. Like so many other cancers, p53 is mutated in over 90% of cancers with BRCA1 mutations - checkpoints further deregulated and DNA replication occurs unguarded. BRCA1 and p53 mutants developed tumors; cells cannot correct the damaged DNA, but they also escape apoptosis. Mutations in BRCA1 cause genomic instability and in p53 allow them to keep growing. Conditional mammary tumor model- inactivate p53 at breast and skin tissues only. Crossed conditional p53 with K14cre transgenic mice; cre recombinase targeted to express only in mammary and skin epithelium. A. Only p53 inactive; B. BRCA1 inactive – no mice developed a mammary tumor in 800 days, most tumors with both inactive Liu et al. (2007)

7 Stöppler. (2016) Myriad Genetics.Increased cell proliferation with unstable genome leads to breast and other cancers ⅛ women will develop breast cancer; only 5-10% of breast cancer caused by BRCA mutations Hereditary Breast and Ovarian Cancer Syndrome - predisposition by germline mutation in BRCA1/2 Autosomal dominant, high penetrance; early onset breast cancer (50-80%) and increased risk of ovarian cancer (30-50%); this results in cancer from loss of heterozygosity Increased sensitivity to genomic stressors, like radiation genetic screening, famous by Angelina Jolie; Having mutated gene increases risk, then options for more regular exams, mastectomy, early detection BRCA1 mutations also found in prostate cancer; men can get breast cancer too, just much lower odds. Also found in pancreatic and colon cancers - environmental factors affect target organ. Stöppler. (2016) Myriad Genetics.

8 Caestecker and Van de Walle. (2012) Sigl et al. (2016)Targeted drug therapy with Olaparib, Denosumab reduces cell proliferation Treatment: PARP inhibitors Its harder to compensate for the loss of inhibition by a tumor suppressor than an oncogene Poly ADP ribose polymerase Cell relies on SSB repair when BRCA1 protein is deficient; PARP is an enzyme for SSB repair → either SSB repair by PARP OR Non-homologous end junction (which is not effective). When PARP is inhibited, SSB repair is not an option → cell death BRCA1 needed for normal cell function; PARPi don’t affect WT BRCA because DSB repair correctly Inhibitors work on BRCT domain mutations = Synthetic lethality, inhibiting two individual pathways resullts in cell death (but not alone) Mastectomy reduces risk by over 90%, salpingo oophrectomy (removal of fallopian tube and ovaries) reduces breast by 50% and ovary by 85% - Estrogen increases growth and spread in most cancers New research about RANK inhibitors on its ligand – paracrine factor on epithelial breast tissue; forms lactating mammary glands; inhibiting RANK ligand reduces proliferation and DNA damage Caestecker and Van de Walle. (2012) Sigl et al. (2016)

9 References Caestecker, Kevin W., and Gerlinde R. Van De Walle. "The Role of BRCA1 in DNA Double-strand Repair: Past and Present." Experimental Cell Research (2013): PDF. Fasano, J, and F. Muggia; Breast cancer arising in a BRCA-mutated background: therapeutic implications from an animal model and drug development. Ann Oncol 2009; 20 (4): Web. Hall, J., M. Lee, B. Newman, J. Morrow, L. Anderson, B. Huey, and M. King. "Linkage of Early-onset Familial Breast Cancer to Chromosome 17q21." Science (1990): Web. Liu, X., H. Holstedge, H. Van Der Gulden, M. Treur-Mulder, J. Zevenhoven, A. Velds, R. Kerkhoven, M. Van Vliet, L. Wessels, J. Peterse, A. Berns, and J. Jonkers. "Somatic Loss of BRCA1 and P53 in Mice Induces Mammary Tumors with Features of Human BRCA1-mutated Basal-like Breast Cancer." Proceedings of the National Academy of Sciences of the United States of America (2007): Web. Maxwell, Kara N., and Susan M. Domchek. "Cancer Treatment According to BRCA1 and BRCA2 Mutations." Nature Reviews Clinical Oncology 9.9 (2012): Web. Miki, Y., J. Swensen, D. Shattuck-Eidens, PA Futreal, K. Harshman, S. Tavtigian, Q. Liu, C. Cochran, LM Bennett, W. Ding, and Al. Et. "A Strong Candidate for the Breast and Ovarian Cancer Susceptibility Gene BRCA1." Science. American Association for the Advancement of Science, 07 Oct Web. "Myriad Genetics | Products & Services | BRACAnalysis." Myriad Genetics. N.p., n.d. Web. Roy, Rohini, Jarin Chun, and Simon N. Powell. "BRCA1 and BRCA2: Different Roles in a Common Pathway of Genome Protection." Nature Reviews Cancer 12.1 (2011): Web. Scully, Ralph, Junjie Chen, Annemieke Plug, Yonghong Xiao, David Weaver, Jean Feunteun, Terry Ashley, and David M. Livingston. "Association of BRCA1 with Rad51 in Mitotic and Meiotic Cells." Cell 88.2 (1997): Web. Sigl, Verena, Kwadwo Owusu-Boaitey, Purna A. Joshi, Anoop Kavirayani, Gerald Wirnsberger, Maria Novatchkova, Ivona Kozieradzki, Daniel Schramek, Nnamdi Edokobi, Jerome Hersl, Aishia Sampson, Ashley Odai-Afotey, Conxi Lazaro, Eva Gonzalez-Suarez, Miguel A. Pujana, For Cimba, Holger Heyn, Enrique Vidal, Jennifer Cruickshank, Hal Berman, Renu Sarao, Melita Ticevic, Iris Uribesalgo, Luigi Tortola, Shuan Rao, Yen Tan, Georg Pfeiler, Eva Yhp Lee, Zsuzsanna Bago-Horvath, Lukas Kenner, Helmuth Popper, Christian Singer, Rama Khokha, Laundette P. Jones, and Josef M. Penninger. "RANKL/RANK Control Brca1 Mutation-driven Mammary Tumors." Cell Research 26.7 (2016): Web. Stöppler, MD Melissa Conrad. "Breast Cancer Awareness Pictures, Symptoms & Treatment." OnHealth. N.p., 12 July Web. Wu, Jiaxue, Lin-Yu Lu, and Xiaochun Yu. "The Role of BRCA1 in DNA Damage Response." Protein & Cell 1.2 (2010): Web. Zhang, Fan, Qiang Fan, Keqin Ren, and Paul R. Andreassen. "PALB2 Functionally Connects the Breast Cancer Susceptibility Proteins BRCA1 and BRCA2." Molecular Cancer Research 7.7 (2009): Web. Zhang, Junran, and Simon N. Powell. "The Role of the BRCA1 Tumor Suppressor in DNA Double-Strand Break Repair." Molecular Cancer Research 3.10 (2005): Web. Zhang, Feng, Jianglin Ma, Jiaxue Wu, Lin Ye, Hong Cai, Bing Xia, and Xiaochun Yu. "PALB2 Links BRCA1 and BRCA2 in the DNA-Damage Response." Current Biology 19.6 (2009): Web. Zheng, Lei, Shang Li, Thomas G. Boyer, and Wen-Hwa Lee. "Lessons Learned from BRCA1 and BRCA2." Oncogene (2000): Web.