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A novel approach for modeling development of breast cancer

Periodic Reporting for period 1 - BRCANCER (A novel approach for modeling development of breast cancer)

Reporting period: 2016-01-04 to 2017-07-03

Inheriting one mutant copy of the BReast CAncer 1 (BRCA1) or BRCA2 gene is linked to a significant increased risk of developing breast, ovarian and other cancers. BRCA1 and BRCA2 mutations account for about 5 to 10 percent of all breast cancers, the most common cancer type in the women worldwide. BRCA1 is involved in various pathways essential for genomic maintenance such as the DNA damage response, X-chromosome inactivation, and cell cycle control. BRCA2 function is largely restricted to DNA recombination and repair processes. Genomic instability caused by loss-of-function mutations in BRCA1 or BRCA2 probably underlies the cancer predisposition, however, the critical mechanisms by which loss or mutagenesis of BRCA1 or BRCA2 leads to tumorigenesis remain unclear. Of the six major breast tumor subtypes, familiar BRCA1 mutant tumors segregate strongly with basal-like cancers and are among the most aggressive. Basal-like tumors account for ~20% of breast cancers and are characterized by triple negativity for estrogen receptor (ER), progesterone receptor (PR) and the human epidermal growth factor receptor 2 (HER2). The absence of ER and HER2 expression precludes the use of endocrine therapy or anti-HER2 treatment and limits systemic treatment to cytotoxic chemotherapy1. For both BRCA1- and BRCA2-dependent cancers, the efficacy of treatments greatly varies between individuals. Together, it highlights the need for development of novel biomarkers, treatment strategies and personalized medicine approaches for breast cancer.

The objectives for the outgoing phase are:
1. To define the sequence of tumor suppressor gene mutations required to induce breast cancer
2. To identify molecular pathways and genetic profiles affected during BRCA1- or BRCA2- dependent breast cancer development

The objectives for the return phase are:
1. To establish relations between in vitro breast tumor organoids, in vivo human-in-mouse xenografts and the original tumor
2. To characterize drug responsiveness of breast tumor organoids derived from different individuals

To address these objectives, I will make use of state-of-the-art breast organoid cultures, CRISPR/Cas9-mediated gene editing and human-in-mouse xenografts (see Table 1 for a detailed explanation of these methodologies).
Work Package 1
The appropriate CDA’s were obtained for using noggin-producing cells generated by the Hubrecht and for using the human breast organoids culture conditions developed by the Hubrecht. The breast organoid model was introduced into the Visvader/Lindeman Lab. Organoids were generated from frozen vials of single cells freshly isolated form human mammary gland tissue. The protocols for passaging and freezing were optimized.

Work package 2
In house (WEHI) lentiviral CRISPR/Cas9 constructs were used for the project. Guides were designed for PTEN, P53, RB, GATA3 and BRCA1 and cloned into the constructs. In the meantime the lentiviral transduction protocol was optimized for human breast organoids. The MCF10A cell line was used to test guide efficiency and the best 2 guides were selected per gene. Then CRISPR/Cas9 was applied on human organoids and knock out lines with different gene combinations were generated in organoids derived from Normals (n>3) and BRCA1 (n>3) mutation carriers. In vitro growth properties were characterized and because of reduced proliferation upon BRCA1 and GATA3 KO these genes were excluded from further in vivo transplantation assays.

Work package 3
CRISPR/Cas9-edited organoids were transplanted in different experiments. Different conditions were tested, including varying cell number, passage number, donor, site (subcutaneously versus mammary fat pad) and combination of mutations (mainly P53/PTEN KO versus P53/PTEN/RB1 KO). In the first round of experiments small fluorescent outgrowths of transplanted cells could be observed, but no tumours were formed. The transplanted glands were harvested 6 months after transplantation. The second round of in vivo experiments is still ongoing and is being continued by a PhD student (J. Whittle) of the Visvader/Lindeman lab.

Work package 4
For the second round of experiments, transplantation conditions were optimized based on results of the first rounds of experiments and now bigger outgrowths/tumours are currently growing. The tumours will be harvested by J. Whittle for further RNA, DNA and histology analysis. Differences between outgrowths of organoids generated from Normal versus BRCA1-/+ donors will be compared.

Work package 5
At WEHI the CRISPR/Cas9-edited organoid lines were transplanted and vivo and in parallel organoids were maintained in vitro. This will allow further analysis of differences between in vivo and in vitro cultures.

Work package 6
At WEHI I have established organoid lines from 10 different triple negative breast cancers samples and they were stored in liquid nitrogen. Some pilot experiments were performed on setting op drug efficacy assays using different chemotherapies and targeted-therapies, but this line was not continued because of lack of time. The Visvader/Lindeman group currently builds on these pilot experiment for further organoid-based drug assays.
The project so far had its expected impart on my personal career: I have obtained new skills in molecular biology, animal work, scientific writing and presenting. The project has created further awareness of the potential of the organoid model in Australia, as I have presented several times at WEHI and on conferences. It also has already generated novel insight in potential of transforming cells intro cancer cells and knowledge in breast cancer biology will grow when the project continues.