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A novel approach for modeling development of breast cancer

Periodic Reporting for period 2 - BRCANCER (A novel approach for modeling development of breast cancer)

Reporting period: 2017-07-04 to 2018-07-03

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).

Conclusions

- It is feasible transform normal cells into tumor cells by knocking out the tumor suppressor genes P53, PTEN and RB
- P53/PTEN/RB-KO lines have increased growth properties in vitro as compared to non-transformed cells
- the clonal diversity during tumor progression in vivo is similar to the clonal diversity in in vitro organoids where tumors originated from
- Both in vivo tumors and in vitro organoids respond to drug treatment
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. During the outgoing phase, NF1 KO was added in addtion to PTEN, P53 and RB.

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 versus P53/PTEN/RB1/NF1). In the first round of experiments small fluorescent outgrowths of transplanted cells could be observed, but no tumours were formed. Result showed that transformation of normal cells into tumour cells with at least 3 mutations is feasible.

Work package 4
Histology of the tumors from the first donor have been fully characterized: tumors were positive for ER and PR, and negative for HER2. Also, next generation sequencing (miSeq) has been performed and results indicated that the clonal diversity is maintained in in vivo tumors, as compared to their in vitro organoids lines.

Work package 5
next generation sequencing (miSeq) has been performed on both the in vitro organoids, as well as on tumors generated from these organoids. Results indicated that the clonal diversity present in the organoids is being maintained during tumor progression in vivo and that clonal restriction in these tumors is limited. In addition, drug treatment has been performed in vivo and in vitro using tamoxifen, and both responded to the treatment as expected.

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.

Dissemination of results:
WEHI Wednesday seminar, March 2016, oral presentation, 200 attendees
Lorne Cancer conference, January 2017, poster presentation, 300 attendees
WEHI stem cells and cancer meeting, May 2017, oral presentation, 50 attendees
WEHI Postgraduate lecture series, March 2017, oral presentation, 200 attendees
Imaging symposium Utrecht, August 2017, oral presentation, 50 attendees
U-talent Masterclass Utrecht, January 2018, oral presentation, 20 attendees
ISSCR Conference Melbourne, July 2018, oral presentation, 300 attendees
Organoids are us symposium Melbourne, July 2018, oral presentation, 150 attendees
This project has generated impact for me, the WEHI Institute, the Hubrecht Institute and the scientific community in general. I have set up the human breast organoids technology at the WEHI in Australia, and the model is being used for new projects unrelated to this proposal. I have returned to the Netherlands with new skills in mouse handling, in vivo transplantations, FACS sorting, cloning, breast cancer research in general, and with many new contacts. This is of great use for me and my current lab / institute. I have presented the work at various national and international meetings and several project-related papers are publishes, accepted for publication, or being prepared.