Redefining the Expression and Function of the Inhibitor of Differentiation 1 in Mammary Gland Development

The accumulation of poorly differentiated cells is a hallmark of breast neoplasia and progression. Thus an understanding of the factors controlling mammary differentiation is critical to a proper understanding of breast tumourigenesis. The Inhibitor of Differentiation 1 (Id1) protein has well documented roles in the control of mammary epithelial differentiation and proliferation in vitro and breast cancer progression in vivo. However, it has not been determined whether Id1 expression is sufficient for the inhibition of mammary epithelial differentiation or the promotion of neoplastic transformation in vivo. We now show that Id1 is not commonly expressed by the luminal mammary epithelia, as previously reported. Generation and analysis of a transgenic mouse model of Id1 overexpression in the mammary gland reveals that Id1 is insufficient for neoplastic progression in virgin animals or to prevent terminal differentiation of the luminal epithelia during pregnancy and lactation. Together, these data demonstrate that there is no luminal cell-autonomous role for Id1 in mammary epithelial cell fate determination, ductal morphogenesis and terminal differentiation

FcRgamma activation regulates inflammation-associated squamous carcinogenesis

Chronically activated leukocytes recruited to premalignant tissues functionally contribute to cancer development; however, mechanisms underlying pro- versus anti-tumor programming of neoplastic tissues by immune cells remain obscure. Using the K14-HPV16 mouse model of squamous carcinogenesis, we report that B cells and humoral immunity foster cancer development by activating Fcgamma receptors (FcgammaRs) on resident and recruited myeloid cells. Stromal accumulation of autoantibodies in premalignant skin, through their interaction with activating FcgammaRs, regulate recruitment, composition, and bioeffector functions of leukocytes in neoplastic tissue, which in turn promote neoplastic progression and subsequent carcinoma development. These findings support a model in which B cells, humoral immunity, and activating FcgammaRs are required for establishing chronic inflammatory programs that promote de novo carcinogenesis

Cryopreservation of human cancers conserves tumour heterogeneity for single-cell multi-omics analysis

Background: High throughput single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for exploring cellular heterogeneity among complex human cancers. scRNA-Seq studies using fresh human surgical tissue are logistically difficult, preclude histopathological triage of samples, and limit the ability to perform batch processing. This hindrance can often introduce technical biases when integrating patient datasets and increase experimental costs. Although tissue preservation methods have been previously explored to address such issues, it is yet to be examined on complex human tissues, such as solid cancers and on high throughput scRNA-Seq platforms. Methods: Using the Chromium 10X platform, we sequenced a total of ~ 120,000 cells from fresh and cryopreserved replicates across three primary breast cancers, two primary prostate cancers and a cutaneous melanoma. We performed detailed analyses between cells from each condition to assess the effects of cryopreservation on cellular heterogeneity, cell quality, clustering and the identification of gene ontologies. In addition, we performed single-cell immunophenotyping using CITE-Seq on a single breast cancer sample cryopreserved as solid tissue fragments. Results: Tumour heterogeneity identified from fresh tissues was largely conserved in cryopreserved replicates. We show that sequencing of single cells prepared from cryopreserved tissue fragments or from cryopreserved cell suspensions is comparable to sequenced cells prepared from fresh tissue, with cryopreserved cell suspensions displaying higher correlations with fresh tissue in gene expression. We showed that cryopreservation had minimal impacts on the results of downstream analyses such as biological pathway enrichment. For some tumours, cryopreservation modestly increased cell stress signatures compared to freshly analysed tissue. Further, we demonstrate the advantage of cryopreserving whole-cells for detecting cell-surface proteins using CITE-Seq, which is impossible using other preservation methods such as single nuclei-sequencing. Conclusions: We show that the viable cryopreservation of human cancers provides high-quality single-cells for multiomics analysis. Our study guides new experimental designs for tissue biobanking for future clinical single-cell RNA sequencing studies

A mutation in the viral sensor 2'-5'-oligoadenylate synthetase 2 causes failure of lactation

We identified a non-synonymous mutation in Oas2 (I405N), a sensor of viral double-stranded RNA, from an ENU-mutagenesis screen designed to discover new genes involved in mammary development. The mutation caused post-partum failure of lactation in healthy mice with otherwise normally developed mammary glands, characterized by greatly reduced milk protein synthesis coupled with epithelial cell death, inhibition of proliferation and a robust interferon response. Expression of mutant but not wild type Oas2 in cultured HC-11 or T47D mammary cells recapitulated the phenotypic and transcriptional effects observed in the mouse. The mutation activates the OAS2 pathway, demonstrated by a 34-fold increase in RNase L activity, and its effects were dependent on expression of RNase L and IRF7, proximal and distal pathway members. This is the first report of a viral recognition pathway regulating lactation.This work was supported by grants from the Congress Directed Medical Research Program (BC995364 and DAMD17-01-1-0241), Cure Cancer Australia Foundation, NHMRC Australia (projects 1047149, Fellowships 1058356, 481310, 1043400), the Australian Research Council Discovery Project (DP110102288), Princeton University, NIH grant 1R01GM110161-01 (AK), Sidney Kimmel Foundation for Cancer Research (AK), Burroughs Wellcome Foundation (AK), Banque Nationale de Paris-Paribas Australia and New Zealand, Mostyn Family Foundation, Cue Clothing Co., Estee Lauder Australia, RT Hall Trust and Fellowships (ECF-13-08 and ECF-16-022) from the National Breast Cancer Foundatio

Proteogenomic analysis of Inhibitor of Differentiation 4 (ID4) in basal-like breast cancer

Funder: National Health and Medical Research Council; doi: http://dx.doi.org/10.13039/501100000925Abstract: Background: Basal-like breast cancer (BLBC) is a poorly characterised, heterogeneous disease. Patients are diagnosed with aggressive, high-grade tumours and often relapse with chemotherapy resistance. Detailed understanding of the molecular underpinnings of this disease is essential to the development of personalised therapeutic strategies. Inhibitor of differentiation 4 (ID4) is a helix-loop-helix transcriptional regulator required for mammary gland development. ID4 is overexpressed in a subset of BLBC patients, associating with a stem-like poor prognosis phenotype, and is necessary for the growth of cell line models of BLBC through unknown mechanisms. Methods: Here, we have defined unique molecular insights into the function of ID4 in BLBC and the related disease high-grade serous ovarian cancer (HGSOC), by combining RIME proteomic analysis, ChIP-seq mapping of genomic binding sites and RNA-seq. Results: These studies reveal novel interactions with DNA damage response proteins, in particular, mediator of DNA damage checkpoint protein 1 (MDC1). Through MDC1, ID4 interacts with other DNA repair proteins (γH2AX and BRCA1) at fragile chromatin sites. ID4 does not affect transcription at these sites, instead binding to chromatin following DNA damage. Analysis of clinical samples demonstrates that ID4 is amplified and overexpressed at a higher frequency in BRCA1-mutant BLBC compared with sporadic BLBC, providing genetic evidence for an interaction between ID4 and DNA damage repair deficiency. Conclusions: These data link the interactions of ID4 with MDC1 to DNA damage repair in the aetiology of BLBC and HGSOC

The role of inhibitor of DNA binding 4 (Id4) in mammary gland development and breast cancer

The inhibitor of DNA binding (Id) proteins Id1-4 are transcriptional regulators that control many cell fate and developmental processes and are often deregulated in cancer. In this dissertation I examine the role of Id proteins in mammary development and neoplasia. Initially the role of Id1 in regulating the immune response to senescent tumour cells was examined, before a more thorough investigation into the role of Id4 during mammary gland development and breast cancer. Prior to the studies described in this thesis, the role for Id4 during mammary gland development had not been investigated and its role in breast cancer was controversial. Id4 expression patterns in the mammary gland were analysed throughout development by immunohistochemistry and the phenotype of Id4 loss was determined using the Id4 knockout mouse. The function of Id4 was further analysed using the normal mouse mammary epithelial cell line Comma-Db. Id4 expression was examined in breast cancer cell lines and a number of cohorts of breast cancer patients. The results presented here show that Id4 is a critical regulator of mammary gland development through its control of differentiation, proliferation and extracellular matrix (ECM) remodelling pathways. Id4 expression is restricted to the myoepithelial cells of the mammary gland and its loss leads to impaired mammary gland development. Overexpression and knockdown studies utilising the Comma-Db cells demonstrated that Id4 inhibited luminal differentiation and was required for cell proliferation. Furthermore Id4 overexpression promoted neoplastic transformation of the normal Comma-Db cell line in vivo. Transcript profiling experiments further demonstrated Id4 regulated a number of ECM remodelling genes. Our results from examining patient cohorts demonstrate that Id4 expression associates with the Her2 and basal-like subtypes of breast cancer and that high expression significantly correlates with improved patient survival. Despite high Id4 expression associating with improved survival, our combined results demonstrate that Id4 nonetheless has the capacity to promote tumourigenesis